Workshop

FTIR Spectroscopy in Microbiological and Medical Diagnostics

Robert Koch-Institute, Berlin October 19-20, 2017

Programme and Abstracts

Workshop

FTIR Spectroscopy in Microbiological and Medical Diagnostics

Robert Koch-Institute, Berlin October 19-20, 2017

Venue and Time

Robert Koch-InstituteNordufer 20, 13353 Berlin, Germany

Registration: October 19, 2017 8:30 – 9:30 Beginning: October 19, 2017 9:30 Ending: October 20, 2017 16:55

Programme

09:30 - 09:40 Lothar H. Wieler (President of the RKI) Opening remarks Session chair: Janina Kneipp

09:40 - 10:00 Bayden R. Wood (Clayton, Australia) A new infrared spectroscopic point-of-care diagnostic for the detection and quantification of pathogens in red blood cells

Thursday, October 19, 2017

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

10:05 - 10:25 Howbeer Muhamadali (Manchester, U.K.) Quantitative differentiation of isotopically labelled Escherichia coli at single cell and community levels via multiple spectroscopy techniques

10:30 - 10:50 Jörg Rau (Stuttgart, Germany) MALDI-TOF MS and FTIR spectroscopy for the identification and characterization of bacteria isolated from elephant and rhinoceros

10:55

- 11:25 Coffee Break Session chair: Malgorzata Baranska

11:25 - 11:45 Ganesh D. Sockalingum (Reims, France) Identification of filamentous fungi by high-throughput FTIR spectroscopy and supervised chemometric methods

11:50 - 12:10 Christoph Krafft (Jena, Germany) Towards translation of Raman spectroscopy for cell identification into clinical laboratories

12:15 - 12:35 Janina Kneipp (Berlin, Germany) Multiphoton excitation and vibrational microspectroscopy of plant tissues

12:40 - 12:55 Volha Shapaval (Ås, Norway) FTIR spectroscopy for analyzing lipids in microbial cells

13:00 - 14:10 Lunch Session chair: Ganesh Sockalingum

14:10 - 14:30 Philip N. Bartlett (Southampton, U.K.) DNA detection and discrimination using electrochemical SERS

14:35 - 14:55 Phil Heraud (Clayton, Australia) Infrared spectroscopy provides new insights in marine science

15:00 - 15:20 Jean-Pierre de Vera (Berlin, Germany) Raman-spectroscopy for life detection on Mars and the Icy Moons in the outer solar system

15:25 - 15:45 Giovanni Longo (Rome, Italy) Movement at the nanoscale to tackle biomedical challenges

15:50 - 16:20 Coffee Break

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

Session chair: Jürgen Schmitt

16:20 - 16:40 Monika Ehling-Schulz (Vienna, Austria) FTIR spectroscopy – Biophotonics meets veterinary medicine

16:45 - 17:00 Kamilla Malek (Krakow, Poland) FTIR spectroscopic imaging using standard and high magnification resolution: to detect inflamed and cancer cells in vitro and ex vivo

17:05 - 17:10 Miriam Unger (Santa Barbara, USA) Latest advancements in nanoscale IR spectroscopy: spatial resolution, speed and spectral range

17:10 - 17:20 Max Eisele (Martinsried, Germany) Exploring micro and nanobiological tissue at the nanoscale using infrared nano-spectroscopy (nano-FTIR)

17:20 - 17:30 Markus Mangold (Zurich, Switzerland) Single-shot microsecond-resolved spectroscopy of the bacteriorhodopsin photo cycle with quantum cascade laser frequency combs

17:30 - 17:40 Otto Hertzberg & Alexander Bauer (Frankfurt/Main, Germany) Mid-IR photothermal deflection spectrometer based on quantum cascade lasers: towards non-invasive glucose measurement

17:40 - 19:30 Poster Session

Poster Session P1 A. Banas (Singapore, Singapore)

Qualitative analysis of human sebum in sebaceous glands in-situ using FTIR microspectroscopy

P2 K. Banas (Singapore, Singapore) Comparison of work-flows for spectral data pre-processing and multivariate statistical analysis by means of open source solutions: orange (Python) and R studio (R language)

P3 G. Bellisola (Frascati, Italy) Infrared analysis of cystic fibrosis (CF) cell models

P4 O. Bibikova (Berlin, Germany) MIR-fiber spectroscopy for tumor sensing: in competition with Raman, NIR-reflection, fluorescence – or in combination?

P5 R. Breitenbach, N. Knabe (Berlin, Germany) Microscopy-based Raman spectroscopy of fungal melanins in a genetically amenable Ascomycete

P6 D. Casagrande Pierantoni (Perugia, Italy) High-contrast Brillouin and Raman micro-spectroscopy for simultaneous mechanical and chemical investigation of microbial biofilms

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

P7 N. Chaudhary (Dublin, Ireland) Evaluation of T-cell activation with Raman microspectroscopy

P8 E. Cordero (Jena, Germany) A compact Raman imaging system for bladder tissue analysis

P9 L. Corte (Perugia, Italy) Merging FTIR and NGS for simultaneous phenotypic and genotypic identification of pathogenic Candida species

P10 M. Dahms (Jena, Germany) Raman micro-spectroscopic identification of Streptococcus pneumoniae differentiated from other Streptococcus species

P11 J. Denbigh (Manchester, U.K.) Synchrotron infrared microspectroscopy as a tool for probing drug-cell interactions in living biological cells

P12 D. Fioretto (Perugia, Italy) Microbial single cell detection with Raman spectroscopy: taxonomic resolution and data accuracy

P13 A. Flack (Reims, France) Vibrational spectroscopy as a high throughput technique for bacteria identification

P14 S. Fornasaro (Trieste, Italy) Surface-enhanced Raman spectroscopy for therapeutic drug monitoring in oncology: a study on sample preparation

P15 C. García-Timermans (Ghent, Belgium) Sample preparation for bacteria identification with Raman spectroscopy

P16 M. Grube (Riga, Latvia) FT-IR microspectroscopy of cancer cells and extracellular vesicles

P17 T. Grunert (Vienna, Austria) Deciphering Staphylococcus aureus surface glycostructures by FTIR spectroscopy

P18 C. Hartmann (Munich, Germany) Modified protein complexes for non-invasive molecular control

P19 Z. Heiner (Berlin, Germany) Surface enhanced hyper Raman spectroscopy for bioapplications

P20 H. M. Heise (Iserlohn, Germany) Silver halide fibers and other materials relevant for attenuated total reflection and transmission infrared spectroscopy: biocompatibility and toxicity testing using fibroblast cells

P21 M. Hermes (Exeter, U.K.) Developing a rapid screening mid-IR imaging method for diagnosis of oesophageal cancer

P22 P. Hoffmann (Jena, Germany) Detection of the cellular uptake and localization of photoCORMs by means of FT-IR micro-spectroscopic imaging

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

P23 A. Jaworska (Warsaw, Poland) PM-IRRAS and AFM studies on modified ssDNA adsorbed on gold

P24 K. Kochan (Clayton, Australia) AFM-IR nanoscale study of cell walls in living bacteria

P25 C. Kratz (Berlin, Germany) Optofluidic platform for enhanced IR microscopic biosensing

P26 F. Küçük Baloğlu (Ankara, Turkey) Investigation of therapeutic effect of palmitoleic acid on obesity induced type 2 diabetes in adipose tissue by Fourier transform infrared microspectroscopy

P27 W. M. Kwiatek (Kraków, Poland) Application of AFM-IR to study human lenses and chromosomes

P28 F. Lauer (Berlin, Germany) Identification of pollen grains in mixtures using hyperspectral MALDI-TOF MS imaging

P29 I. M. Le-Deygen (Moscow, Russia) Interaction of magnetic nanorods coated by dopamine with anionic liposomes as revealed by FTIR spectroscopy

P30 C. A. Lima (São Paulo, Brazil) Infrared spectroscopy determining the biochemical changes in premalignant skin lesions submitted to photodynamic therapy

P31 X.-Y. Liu (Jena, Germany) Confocal Raman imaging integrated with non-negative matrix factorization analysis on spatiotemporal distribution of major components in biofilm

P32 B. Lorenz (Jena, Germany) Preparation of blood samples for Raman microspectroscopy on single bacteria cells

P33 J. Mathurin (Orsay, France) Label-free imaging to characterize new antibiotics carriers by IR nanospectroscopy

P34 A. S. Mondol (Jena, Germany) Development of Raman platform for single cell analysis

P35 O. Morgaienko (Munich, Germany) Visualization of pollutant degrading bacteria via bioorthogonal noncanonical amino acid tagging coupled to surface-enhanced Raman scattering

P36 C. Paluszkiewicz (Kraków, Poland) Studies of cancerous tissues composition using FTIR and Raman microspectroscopy methods

P37 L. Quaroni (Kraków, Poland) AFM-IR spectromicroscopy and imaging of vesicles, micelles, organelles and cytoskeletal structures in fibroblasts

P38 L. Quaroni (Kraków, Poland) Nanoscale mid-infrared spectroscopic and imaging studies of single phospholipid bilayers as models of biological membranes

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

P39 L. Roscini (Perugia, Italy) “Checker-FTIR”: applying FTIR to checker board assays for drug mixtures inhibition tests

P40 A. Rüther (Clayton, Australia) A multimodel approach to Babesia bovis diagnosis

P41 I. W. Schie (Jena, Germany) High-throughput Raman spectroscopy of single cells

P42 V. Shapaval (Ås, Norway) FTIR spectroscopy for HT-screening and monitoring of single cell oil production

P43 T. Shaykhutdinov (Berlin, Germany) IR nanopolarimetry: anisotropy in biomolecular assemblies and thin biofilms

P44 K. Shvirksts (Riga, Latvia) Single factor stress response studies of mcf-7 breast cancer cells by FTIR spectroscopy

P45 J. Solheim (Ås, Norway) Fast resonant Mie-scatter correction algorithm: parameter choice and validation

P46 L. Sykora (Munich, Germany) ATR-FTIR microplate reader and micromachined ATR silicon crystals

P47 R. Weiss (Munich, Germany) Raman microspectroscopy for non-invasive, three-dimensional analysis of biofilms

P48 C. Wichmann (Jena, Germany) Influence of CO2-concentration on Raman spectra of bacteria

P49 W. Yang (Jena, Germany) Fiber optic probe-based Raman imaging using positional tracking

P50 I. Zeise (Berlin, Germany) Analysis of plant tissues using vibrational and other spectroscopic methods and multivariate approaches

P51 Vesna Živanović (Berlin, Germany) Comprehensive vibrational characterization of the interaction of liposomes and gold nanoparticles

P52 C. Fígoli (LaPlata, Argentina) Vibrational micro- and nanospectroscopy of Burkholderia contaminans biofilms

P53 C. Beleites (Wölfersheim,, Germany) Experimental designs for comparing variance contributions in nested data

P54 A.R. Walther (Odense, Denmark) Investigating the uptake and response of hMSC cells exposed to Falcarindiol

P56 S. Diehn (Berlin, Germany) Hierarchical classification of variations in grass pollen quality using MALDI-TOF MS

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

Session chair: Achim Kohler

09:00 - 09:20 Michael Wagner (Vienna, Austria) Functional analyses and targeted single cell genomics of microbes by Raman microspectroscopy

09:25 - 09:45 Matthew J. Baker (Glasgow, U.K.) Developing spectroscopic processes for the detection of bacteria: environmental, surface deposited and high throughput

09:50 - 10:10 Richard A. Dluhy (Birmingham, USA) Direct characterization of stored red blood cells using Raman spectroscopy

10:15 - 10:35 Luísa Peixe (Porto, Portugal) Application of FTIR and MALDI-TOF MS for bacterial typing: a current standpoint

10:40 - 11:10 Coffee Break Session chair: Max Diem

11:10 - 11:30 Malgorzata Baranska (Kraków, Poland) Primary cells vs. cell lines: in vitro experiments and spectroscopic analysis

11:35 - 11:50 Helene Oberreuter (Stuttgart, Germany) Cross-border Salmonellosis outbreak linked to fresh sprouts – analysis of Salmonella bovismorbificans isolates by FTIR-spectroscopy

11:55 - 12:15 Ariane Deniset-Besseau (Orsay, France) Advanced IR nanospectroscopy to study lipids bodies in micro-organisms: toward a better understanding of metabolic pathways at stake

12:20 - 12:35 Curtis Marcott (Athens, USA) Non-contact methodology for obtaining submicron IR spectra and images of cells and tissue

12:40 - 12:55 Petra Rösch (Jena, Germany) Cultivation-free identification of lung bacteria by means of Raman spectroscopy

Friday, October 20, 2017

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

13:00 - 14:00 Lunch Session chair: Ariane Deniset-Besseau

14:00 - 14:20 Achim Kohler (Ås, Norway) Model-based pre-processing for estimating scattering and absorption in infrared spectroscopy

14:25 - 14:45 Cecilia Figoli (La Plata, Argentina) Vibrational micro- and nanospectroscopy of Burkholderia contaminans biofilms

14:50 - 15:05 N. P. Ivleva (Munich, Germany) Applicability of SERS in combination with stable isotope approach for characterization of microorganisms at single cell level

15:10 - 15:40 Coffee Break Session chair: Bayden Wood

15:40 - 15:55 Gianluigi Cardinali (Perugia, Italy) FT-IR applied to microbial identification and stress response quantification: a preliminary balance and new perspectives

16:00 - 16:15 Michael Hermes (Exeter, U.K.) Advancing mid infrared imaging technology: the Mid-TECH project

16:20 - 16:40 Max Diem (Boston, USA) Results from a large-scale lung cancer spectral histopathology (SHP) study

16:45 - 16:55 Final Discussion, Concluding Remarks

Workshop ″FTIR Spectroscopy in Microbiological and Medical Diagnostics″

Aim

The 2017 Workshop will continue the tradition of highlighting every two years the relevant fields of applications of biomedical vibrational spectroscopy and will bring together scientists using infrared and Raman spectroscopic techniques for the characterization and differentiation of intact microbial, plant, animal or human cells to promote exchange of ideas, experiences, and practical problem solutions. Following the lines of our last workshops in Berlin, major points of discussion will be the progress in vibrational spectroscopic research, recent applications in various fields of microbiology, bio-medicine and new technological developments.

Organization Dr. P. Lasch, RKI Berlin, Phone: +49 30 18754 2259, E-Mail: laschp@rki.de, Dr. J. Schmitt, Synthon GmbH, E-Mail: schmitt@synthon-analytics.de and The International Society for Clinical Spectroscopy (CLIRSPEC)

Contact Address Peter Lasch, Robert Koch-Institute, ZBS 6 Phone.: +49-30-18754 2259 Nordufer 20, 13353 Berlin, Germany E-Mail: laschp@rki.de

Sponsoring Financial and technical support came from the Robert Koch-Institute

Abstracts of the lectures

A New Infrared Spectroscopic Point-of-care Diagnostic for the Detection and Quantification of Pathogens in Red Blood Cells

Bayden R. Wood, Phil Heraud, Anja Rüther, Brian Cooke, David Perez-Guaita

Centre for Biospectroscopy, Monash University, Wellington Rd. Clayton

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy, in combination with advanced computational modelling, offers tremendous potential for simultaneous, point-of-care diagnosis of multiple infectious diseases [1]. We have demonstrated the potential of the technique to detect parasite concentrations of 1/100,000 from packed red blood cells [2]. More recently we demonstrated how the approach can be used to simultaneously quantify malaria parasitemia, glucose and urea levels from a dried whole blood spot on a glass slide [3]. The ATR-FTIR approach is robust making it an ideal technology for screening of humans and other animals in any setting from remote regions of the developing world to hospital pathology labs or to mass screening in the modern clinical laboratory. ATR spectroscopy relies on detecting the molecular phenotype of the pathogen directly, and can discern malaria and Babesia parasites in red blood cells. Like malaria, Babesia is an Apicomplexan parasite that causes the disease known as Babesiosis, which severely affects cattle. We have developed a methodology that utilises lysis and concentration to isolate and purify malaria and babesia pathogens prior to ATR-FTIR analysis. The talk will focus on the application of the technology to malaria diagnosis in the field and provide preliminary results on the detection and quantification of pathogens that cause Babesiosis in cattle. References [1] F. Martin, M. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. Butler, K. Dorling, P. Fielden, S. Fogarty, N.

Fullwood, C. Hughes, K. Heys, P. Lasch, P. Martin-Hirsch, B. Obinaju, G. Sockalingum, J. Sule-Suso, R. Strong, M. Walsh, B. R. Wood,and P. Gardner, Nature Protocols 9, 1771–1791 (2014).

[2] A. Khoshmanesh, M. W. A. Dixon, S. Kenny, L. Tilley, D. McNaughton, B. R Wood, “Detection and Quantification of Early-Stage Malaria Parasites in Laboratory Infected Erythrocytes by Attenuated Total Reflectance Infrared Spectroscopy and Multivariate Analysis”, Anal. Chem. 86, 4379–4386 (2014).

[3] S. Roy, D. Perez-Guaita, D.W. Andrew, J.S. Richards, D. McNaughton, “Simultaneous ATR-FTIR Based Determination of Malaria Parasitemia, Glucose and Urea in Whole Blood Dried onto a Glass Slide”, Analytical Chemistry 89 (10), 5238-5245.

 

Quantitative Differentiation of Isotopically Labelled E. coli at Single Cell and Community Levels via Multiple Spectroscopy Techniques

Howbeer Muhamadali, Malama Chisanga and Royston Goodacre

School of Chemistry, Manchester Institute of Biotechnology, University of Manchester,

Manchester, UK The contribution of microbially-mediated bioprocesses toward the maintenance of life on Earth is well-evidenced and undeniable. Though the systematic analysis and understandingof these microbes in situcould at present be considered as a bottleneck. This is because most analytical methods require culturing these microorganisms to suitable biomass levels sothat their phenotype can be measured. The development of new culture-independent strategies such as stable isotope probing (SIP), coupled with molecular biology, has been a breakthrough towards linking gene to function, whilst circumventing in vitro culturing. During the past two decades the application of spectroscopy techniques combined with SIP, has emerged as a very promising approach for bacterial characterisation.Thus, in this study we have employed Fourier transform infrared spectroscopy, Raman and surface enhanced Raman scattering (SERS) combined with SIP to demonstrate the quantitative labelling and differentiation of E. coli cells grown in defined medium, withdifferent ratios and combinations of 13C/12C glucose and 15N/14N ammonium chloride, at both single-cell and community levels. It is our hope that our results demonstrate the potential of this combined analytical approach toward a greater understanding of microorganisms, and which may wellhave the capacity to open up an exciting new perspective on the study of living microbes in situ.  

MALDI-TOF MS and FT-IR Spectroscopy for the Identification and Characterization of Bacteria Isolated from Elephant and Rhinoceros

Jörg Rau1, Tobias Eisenberg2,3

  

1 Chemisches und Veterinäruntersuchungsamt Stuttgart (CVUAS), Schaflandstr. 3/2, 70736 Fellbach, Germany; joerg.rau@cvuas.bwl.de

2 Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität Gießen 3 Landesbetrieb Hessisches Landeslabor, Gießen

Samples from zoo animals can pose particular diagnostic challenges. For the identification of microorganisms isolated from these samples, biochemical and biomolecular methods are increasingly supplemented by spectroscopic methods. In our laboratories, MALDI-TOF mass spectrometry (MS) is routinely used for genus and species identifications. For further differentiation below the species level, Fourier-Transform-infrared spectroscopy (FT-IR) is utilized. In the first example, we describe the comparative analysis von 23 Streptococcus agalactiae isolates from Asian and African elephants in four German zoos. The samples originated from infected wounds as well as from chronical pododermatitids in these animals [1]. Especially FT-IR allowed for the fast differentiation of various infection clusters, which would otherwise only be detectable through laborious biomolecular methods. In the second example, the deviating MALDI-TOF MS and FT-IR spectra from a bacterial isolate from an Indian rhinoceros (Rhinoceros unicornis) indicated the presence of a presumably undescribed species. Extensive analyses, coordinated by the Justus-Liebig-Universität Gießen, have by now leaded to the description of Arcanobacterium wilhelmae, named after the zoological and botanical garden in Stuttgart [2]. Both examples demonstrate that the depth of the differentiation achieved by the combination of MALDI-TOF MS and FT-IR are a gain for veterinary diagnostics. References [1] T. Eisenberg, J. Rau et al., “Streptococcus agalactiae in elephants – a comparative study – with

isolates from human, zoo animal and livestock origin”, Vet. Microbiol. 204, 141-150 (2017). [2] O. Sammra, J. Rau et al., “Arcanobacterium wilhelmae sp. nov. isolated from the genital tract of a rhinoceros

(Rhinoceros unicornis)”, IJSEM online ahead of print (2017).

Identification of Filamentous Fungi by High-throughput FTIR Spectroscopy and Supervised Chemometric Methods

A. Lecellier1, V. Gaydou1, J. Mounier2, D. Toubas1,3, G. D. Sockalingum1*

1Université de Reims Champagne-Ardenne, UFR de Pharmacie,MéDIAN-Biophotonique et Technologies pour la Santé, CNRS UMR 7369,51 rue Cognacq-Jay, 51096 Reims Cedex,

France 2Université de Brest, EA3882 LaboratoireUniversitaire de

BiodiversitéetEcologieMicrobienne, IBSAM, ESIAB, Technopôle Brest Iroise, 29280, Plouzané, France

3Laboratoire de ParasitologieMycologie, CHU de Reims, Université Reims Champagne Ardenne, HôpitalMaison Blanche, Reims, 51092, France

*ganesh.sockalingum@univ-reims.fr

Abstract Mold contaminants represent a major problem in various areas such as food and agriculture, pharmaceutics, cosmetics and health. Currently, mold identification is based either on phenotypic characteristics, requiring an expertise and can lack accuracy, or on molecular methods, which are quite expensive and fastidious. In this context, the objective was to develop a simple and standardized protocol using FTIR spectroscopy combined with a chemometric analysis, allowing implementation of an alternative method for rapid identification of molds. In total, 486 fungal strains (45 genera and 140 species) were analysed using a high-throughput FTIR spectrometer. Partial Least Squares Discriminant Analysis (PLS-DA), a supervised chemometrics method, was evaluatedfor the identification processusing the spectral ranges 3200-2800 and 1800-800 cm-1. Using 288 strains, different calibration models were constructed in cascade and following the current taxonomy, from the subphylum to the species level. Blind prediction of spectra from 105 strains at the genus and species levels was achieved at 99.17 % and 92.3% respectively. Since sufficient mycelial biomass can be obtained at 48h culture and sample preparation involved a simple protocol, FTIR spectroscopy combined with PLS-DA represents a very rapid and cost effective method, which could be particularly attractive for the identification of molds at the industrial level. The results obtained places FTIR spectroscopy among the avant-garde promising analytical approaches, with high discriminant power and identification capacity, compared to conventional techniques. References [1] A. Lecellier et al., “Differentiation and identification of filamentous fungi by high-throughput FTIR

spectroscopic analysis of mycelia”, Int J Food Microbiol. 168-169, 32-4 (2014). [2] A. Lecellier et al., “Implementation of an FTIR spectral library of 486 filamentous fungi strains for rapid

identification of molds”, Food Microbiology 45 (Pt. A), 126-134 (2014). [3] V. Gaydou et al., “Assessing the discrimination potential of linear and non-linear supervised chemometric

methods on a filamentous fungi FTIR spectral database”, Anal. Methods 7, 766-778 ( 2015).

Towards Translation of Raman Spectroscopy for Cell Identification into Clinical Laboratories

C. Krafft1, R. Kiselev1, I. Schie1, M. Hassoun1, J.H. Clement2, J. Popp1,3

1 Institute of Photonic Technology, Jena, Germany

2 Department of Internal Medicine II, University Hospital Jena, Germany 3 Institute of Physical Chemistry & Abbe Center of Photonics, University Jena, Germany Raman spectroscopy has been successfully applied in the research laboratories to identify

single cells. Goals of our recent work were to apply Raman-based techniques for distinction of white blood cells and tumor cells and translate these approaches into clinical laboratories. Progress in the context of sample handling procedures, sample throughput and automation will be presented to achieve these goals.

Microhole arrays in cartridges and microfluidic chips were developed for improved data handling. Microhole arrays enable to load single cells at defined positions in a reversible way [1]. The strategy in the project was to immobilize ca. 200,000 cells onto the chip, select a subset of candidate cells by rapid optical prescreening, collect a Raman spectrum of each candidate cell, and pick positive identified tumor cells by a robotic arm with a micropipette for subsequent biochemical assays. The second approach starts with in vivo selection of EpCAM positive tumor cells by a functionalized wire. After detachment of the cells from the wire, they are injected into a microfluidic chip where a membrane separates the cells from medium. Cells sediment onto a quartz window for Raman microscopy in an inverted geometry [2]. After enumeration of tumor cells, all cells are lysed in the chip and the lysate is analyzed by nucleic acids based biochemical assays. A lab-on-chip device was developed to collect SERS spectra of cell lysates. The generation of 80 nl droplets containing lysate, silver nanoparticles and KCl as activation agent gives reproducible SERS spectra which were used to distinguish three leukemia cells lines with classification accuracies above 99.5%. Gold nanoparticles were functionalized with antibodies and SERS active reporter molecules to target tumor cells. These nanoparticles were mixed with leukocytes, tumor cells and polystyrene beads and injected into a microfluidic chip. Tumor cells were detected in continuous flow at 50 ms exposure time without trapping [3]. Another way to increase the Raman signals of cells by ca. one order of magnitude is to increase the width of the entrance slit of the spectrograph. Although the spectral resolution decreased from 8 to 48 cm-1 with wider slits, it has been demonstrated that the classification accuracy did not significantly change [4]. Both signal enhancement effects can be used to shorten exposure times and increase throughput. A graphical user interface for a simplified instrument control and a database for automatic storage of all experimental were developed based on open source software. The performance was demonstrated for cell identification using different Raman systems with 785 and 660 nm laser excitation at two clinical laboratories.

Acknowledgements: This work was supported by the European Union within the FP7 collaborative project CanDo (610472), by the BMBF within the project RamanCTC (13N12685 and 13N12686) and by the Leibniz Association through the project Hyperam (SAW-2016-IPHT-2). References [1] U. Neugebauer, C. Kurz, T. Bocklitz, T. Berger, T. Velten, J.H. Clement, C. Krafft, J. Popp,

Micromachines 5, 204-215 (2014). [2] R. Kiselev, I.W. Schie, S. Askrabic, C. Krafft, J. Popp, Biomed. Spectrosc. Imag. 5, 115-127 (2016). [3] I. Freitag, C. Beleites, S. Dochow, J. Clement, C. Krafft, J. Popp, Analyst (2016). [4] I.W. Schie, C. Krafft, J. Popp, J. Biophoton. (2016).

Multiphoton Excitation and Vibrational Microspectroscopy of Plant Tissues

Janina Kneipp

Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin

If a sample is excited with laser light, different physical processes can be used quasi-simultaneously for its optical and spectroscopic characterization. Depending on the responsible process, a great variety of information about a complex biological sample is obtained. Two-photon excited microscopy and also spectroscopy have been gaining increasing attention, which has at least two major reasons: i) In general, the simultaneous interaction of two photons with matter follows different selection rules, therefore two-photon excited processes can deliver spectroscopic information complementary to that attainable in a one-photon excited process. ii) Non-linear excitation offers several methodological advantages over one-photon excitation, particularly for the studies of biological objects, mainly related to its lower-energy excitation and the strong confinement of the excitation volumes [1]. Here, we discuss the combined application of different multi-photon processes in the same microspectroscopic set-up: Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG), and two-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissue sections. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, non-resonant as well as pre-resonant Raman microscopy reveals details about the distribution and composition of the major cell wall constituents. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, two-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging, and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues [2].

Figure 1. Molecular energy level scheme of different two-photon and one-photon processes that can be

combined for multimodal imaging of plant tissues.

Acknowledgement. I am grateful for the fruitful discussion and collaboration with Rivka Elbaum (Hebrew University of Jerusalem, Israel) and her group, who provided Sorghum seeds for this work, and to Peter Lasch (RKI) for providing CytoSpec. The work has been financially supported by ERC grant no 259432 MULTIBIOPHOT, Einstein Foundation Berlin (A-2011-77), DFG GSC 1013 SALSA, and a Chemiefonds Fellowship.

References [1] F. Madzharova, Z. Heiner, J. Kneipp, ChemSoc Rev 46, 3980-3999 (2017). [2] Z. Heiner, I. Zeise, R. Elbaum, J. Kneipp, Manuscript submitted (2017).

FTIR Spectroscopy for Analyzing Lipids in Microbial Cells

V. Shapaval1, G. Kosa1, B. Zimmermann1, V. Tafintseva1, K. Lilland1, K. Forfang1, N. K. Afseth2, A. Kohler1

1 The Faculty of Science and Technology, NMBU, Ås, Norway

2Nofima Mat, Osloveien 1, 1430 Ås, Norway

Recent trends in biotechnology have advanced microbial lipids to one of the main alternative sources of lipids for human, animal and industrial use. In microbial cells, lipids play important roles as structural components of membranes, they provide storage material for excess carbon and they represent the main signaling molecules to mediate the outer signals. Thus, it is of high importance to analyze the structures and profiles of microbial lipids and their metabolites in order to elucidate their functions in microbial cells and to select suitable strains for production of certain lipid classes. FTIR spectroscopy provide a precise biochemical fingerprint of microbial cells and therefore used frequently for biochemical characterization. Recently, FTIR spectroscopy was introduced as a rapid, non-destructive approach to predict and analyze lipids in oleaginous microorganisms [1]. In the present study, we present a high-throughput approach based on FTIR spectroscopy and micro-cultivation in a Microtiter Plate System (MTPS) for the prediction of total lipid content and lipid profiles (saturated, monounsaturated and polyunsaturated fats acids) in microbial cells [2]. The prediction for different taxonomic groups (order, family, genera and species) was investigated and validity was checked. We will further demonstrate an example where the FTIR spectroscopy high-throughput system is used for monitoring lipid extraction processes [3].

Figure 1. High-throughput FTIR-based approach for prediction of lipids in microbes.

References [1] V. Shapaval, N.K. Afseth, G. Vogt, A. Kohler, “Fourier Transform Infrared Spectroscopy for the prediction

of fatty acid profiles in Mucor fungi in media with different carbon sources”, Microbial Cell Factories, (4), 13, 86 (2014).

[2] G.Kosa, A. Kohler, V. Tafintseva, B. Zimmerman, K. Forfang, D. Tzimorotas, N.K. Afseth, K.S. Vuoristo, S.J. Horn, J. Mounier, V. Shapaval, “Duetz Microtiter plate system combined with FTIR spectroscopy for the screening of oleaginous fungi and high-throughput monitoring of lipogenesis”, Microbial Cell Factories 2017(16):101, DOI: 10.1186/s12934-017-0716-7.

[3] K. Forfang, B. Zimmermann, G. Kosa, A. Kohler, V. Shapaval, “Evaluating lipid extraction methods and monitoring their efficiency in oleaginous fungi by FTIR spectroscopy”, PLoS One. 2017; 12(1), DOI: e0170611.

DNA Detection and Discrimination Using Electrochemical SERS

Philip N. Bartlett

Chemistry, University of Southampton, Southampton, SO17 1BJ, UK

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. In this lecture I will describe the use electrochemically driven denaturation assays (E-melting) monitored by SERS to detect and discriminate DNA. The method relies on the high surface enhancement at nanostructured gold electrode surfaces to give very sensitive detection of immobilised dsDNA and to follow the denaturation of the dsDNA as the electrode is swept to negative potentials [1-4]. Using this technique we can discriminate short tandem repeats (STRs) [5], single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from bacterial DNA [6], and discriminate strains within genetically highly similar bacteria DNA using amplicons containing Variable Number Tandem Repeats (VNTRs) [7]. References [1.] S. Mahajan, J. Richardson, T. Brown, P. N. Bartlett, J. Am. Chem. Soc. 130, 15589-15601 (2008). [2.] R. P. Johnson, R. Gao, T. Brown, P. N. Bartlett, Bioelectrochem. 85, 7-13 (2012). [3.] R. P. Johnson, J. A. Richardson, T. Brown, P. N. Bartlett, J. Am. Chem. Soc. 134, 14099−14107 (2012). [4.] R. P. Johnson, N. Gale, J. A. Richardson, T. Brown and P. N. Bartlett, Chem. Sci. 4, 1625-1632 (2013). [5.] D. K. Corrigan, N. Gale, T. Brown and P. N. Bartlett, Angew. Chemie, Int. Edn. 49, 5917-5920 (2010). [6.] E. Papadopoulou, S. A. Goodchild, D. W. Cleary, S. A. Weller, N. Gale, M. R. Stubberfield, T. Brown,

P. N. Bartlett, Anal. Chem. 87, 1605−1612 (2015). [7.] E. Papadopoulou, N. Gale, S. A. Goodchild, D. W. Cleary, S. A. Weller, T. Brown, P. N. Bartlett, Chem.

Sci. 6, 1846–1852 (2015).

Infrared Spectroscopy Provides New Insights in Marine Science

Philip Heraud1,2, Katherina Petrou3, John Beardall4

1Department of Microbiology and theBiomedical Discovery Institute, Monash University, Clayton, Victoria, Australia.

2Centre for Biospectroscopy, Monash University, Clayton, Victoria, Australia. 3School of Life Sciences, University of Technology Sydney, Broadway, New South Wales,

2007, Australia. 4 School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia

Marine phytoplankton are extremely important to the planetary ecosystem fixing

~ 50 Tg of inorganic carbon into organic matter providing the basis for marine food webs. Work over the past decade has seen the introduction of vibrational spectroscopy into marine oceanography focused on the study of phytoplankton. It has been shown that infrared spectroscopy can rapidly assess changes in the macromolecular composition of phytoplankton cells during massive ocean phytoplankton bloom events [1] and can be used to predict the productivity of the phytoplankton using models developed using infrared spectroscopy in a “snapshot” [2]. In recent work we have been using infrared spectroscopy to understand the nutrient changes in the coral phytoplankton endosymbionts brought about by rising temperature. This approach allows nutrient exchange to be understood at the single cell level in situ within the corals, providing a unique insight into subtle changes leading to a breakdown in the relationship between plant and animal symbionts leading to coral bleaching.

Despite decades of research motivated by an increasing frequency of local and global scale bleaching events, the underlying biochemical mechanism that leads to coral bleaching remains poorly understood. We used synchrotron-based FTIR microspectroscopy to examine the macromolecular composition of symbiotic (in hospite – encased in animal cell) and expelled Symbiodinium microalgalcells under healthy conditions and during a bleaching response. The most significant change in the macromolecular profile of thermally stressed expelled cells compared with the in–hospite controls involved the relative concentration of saturated fatty acids and lipids, suggesting either an up-regulation of lipids by the symbiont or reduced translocation of lipids to the host. These data strongly indicate the involvement of changes to lipid production and/or storage by the symbiont in the expulsion and bleaching process. References [1.] O. Sackett, L. Armand,J. Beardall, R. Hill, M. Doblin, C. Connelly, J. Howes, B. Stuart, P. Ralph,

P. Heraud, Biogeosciences 11, 5795-5808 (2014). [2.] O. Sackett, K. Petrou, B., R. Hill, M. Doblin, J. Beardall, P. Ralph, P. Heraud, The ISME Journal 10, 416-

426 (2016).

Raman-Spectroscopy for Life Detection on Mars and the Icy Moons in the Outer Solar System

Jean-Pierre de Vera and the BIOMEX and BIOSIGN-team

German Aerospace Center (DLR), Institute of Planetary Research, Astrobiological

Laboratories, Rutherfordstr. 2, 12489 Berlin

For the existence of life in the Solar System liquid water is required. Promising targets include Mars and the icy moons, Europa and Enceladus. Finding evidence of life within these potentially habitable environments is dependent on finding unique biosignatures that can be used as irrefutable evidence of life. The main aim of the two ESA space experiments BIOMEX [1] and BioSigN is to support future exploration missions to Mars, Enceladus and Europa using a set of exposure experiments to identify feasible organic biosignatures. There are two specific objectives relevant to this aim: 1) analyse the extent to which selected organisms can survive conditions of space and simulated planetary conditions; 2) analyse the stability and degradation of biosignatures in Low Earth Orbit (LEO) and simulated planetary conditions. BIOMEX and BioSigN are using organisms that have previously been isolated from Mars- and sub-surface icy moon analogue sites like Antarctica and the deep sea. The major output of these experiments is a database of spectra, obtained by mainly Raman spectroscopy of organic biosignatures that are detectable after exposure to LEO and simulated planetary conditions. This database will give insight into the stability of biomolecules under different environmental conditions and the value and pitfalls of using the specified instrumentation for life detection missions. Here we will present the rationale behind BIOMEX as well as BioSigN and some results. References [1] J.-P. de Vera, U. Boettger, R. de la Torre Noetzel et al.,Planetary and Space Science 74 (1), 103-110 (2012).

Movement at the Nanoscale to Tackle Biomedical Challenges

Giovanni Longo1, Sandor Kasas2, Marco Girasole1

1 Istituto di Struttura della Materia – Consiglio Nazionale delle Ricerche, Via del Fosso del Cavaliere 100 – 00133 Rome, Italy

2 EPFL – FSB – LPMV, Route de la Sorge – 1015 Lausanne, Switzerland

Movement is life. Studying living biological systems and their nanoscale movements we can achieve a novel insight in their metabolic status and in how they react to external stimuli.

To investigate these movements, we have developed a novel nanoscale sensor, called the nanomotion sensor, which we applied to characterize the innate correlation between life and movement.

Due to its sensitivity, the nanomotion sensor can be used to study bacterial species [1], yeasts and fungi and their response to drugs as well as to chemical or physical stimuli.We will show how the fast response of the sensor, leads to exciting applications in the medical practice, with evident advantages for patients care. For instance, by combining it with rapid isolation of bacteria from clinical samples, we have optimized a protocol to produce a complete characterization of a bacterial infection directly from a clinical source [2].

We will discuss how the extremely high sensitivity of this system can be applied to other systems, including from the study of conformational changes in proteins and protein complexes [3].

Finally, we will present the latest results in the nanomotion characterization of single mammalian cells. As an example, we monitored neurons exposed to amyloid proteins, demonstrating at the single cell level the effect of the different protein aggregation forms [4].

We have also applied this technique to achieve a rapid characterization of theresponse of cancer cells to anti-tumoral drugs, with evident impact in the field of oncology. In fact, just as in the case of the characterization of bacteria, by using the nanomotion detector we determined the susceptibility to a particular therapeutic option for a given cancer in a time-range of hours.

In very general terms, all these pioneering results indicate that there is a close correlation between movement and life and that a sensor capable of transducing these movements can deliver a new point of view in the analysis of living systems and allow a new means to characterize the metabolic activity. This has also led us to propose this nanomotion sensor as an innovative technique to detect life in extreme environments [5]. References [1] Longo et al., Nat. Nanotech. 8, 522-526 (2013). [2] Longo et al., Clin. Microbiol. Infect. 23, 400-405 (2017). [3] Alonso-Sarduy et al., PLoS ONE 9, e103674 (2014). [4] Ruggeri et al., Cell Death Disc., 3 17053 (2017). [5] Kasas et al., PNAS 112 (2), 378–381 (2015).

Figure 1.Setup of a nanomotion experiment. Depictionoftypicalbacteriaorsinglecellsanalyses.

FTIR Spectroscopy – Biophotonics Meets Veterinary Medicine

Monika Ehling-Schulz

Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria

Due to is high discriminatory power and high throughput capacities FTIR spectroscopy has become a well-established biophotonic method in several fields of microbiology, especially in clinical and food microbiology diagnostics. However, in contrast to food microbiology and human clinical microbiology, the potential of FTIR spectroscopy in veterinary medicine is far less explored. Thus, we aimed to assess the suitability of chemometric assisted FTIR spectroscopy for veterinary medicine related applications, in diagnostics and beyond.

Recently, we successfully applied chemometric assisted FTIR to decipherhostgenotype related imprints on bacterial metabolism and to visualize bacterialmemory effects[1,2]. Furthermore, we could show that FTIR spectroscopy is not only a cost-effective powerful tool to track bacteria from ‘farm to fork’ but also allows to link host health status with certain bacterial biotypes[3-5]. Currently, we place special emphasize on exploring the potential of FTIR spectroscopy for studying host pathogen interactions, monitoring within host adaptation processes and analyzing fluctuations in host microbial communities. Results from recent and ongoing research projects will be presented. References [1] T. Grunert et al., “Deciphering host genotype-specific impacts on the metabolic fingerprint of Listeria

monocytogenes by FTIR spectroscopy”, PLOS ONE, 9(12): e115959 (2014). [2] E. L. Sassu et al., “Host-pathogen interplay at primary infection sites in pigs challenged with

Actinobacilluspleuropneumoniae”, BMC Vet. Res. 3, 64 (2017). [3] J. Kümmel et al., “Staphylococcus aureus entrance into the dairy chain: Tracking S. aureus from dairy cow to

cheese”, Front. Microbiol. 7, 1603 (2016). [4] K. Wagener et al., “Dynamics of uterine infections with Escherichia coli, Streptococcus uberis and

Trueperella pyogenes in postpartum dairy cows and their association with clinical endometritis”, Vet. J. 202, 527-532 (2014).

[5] K. Wagener et al., “Diversity and health status specific fluctuations of intrauterine microbial communities in postpartum dairycows”, Vet. Microbiol. 175, 286–293 (2015).

FTR

Ewelin

aFacu

bJa

cCent

I

lines astype α. introducWe choumbilicstudy winflammof markexaminecellular transflecinflameinflamedetectiosimilar from thdetectioidentificof diffe(Fig. 1 I

The A(MB: DE Referenc[1] E. W

Anal [2] K. C

K. M.

TIR Spectesolution:

na WiercigKatarzyn

ulty of Chemagiellonian C

ter for Medi

In this works models to

For this pced by the Aose lung vaal vein end

was to ascmation is spkers for celled to what

compartmction) affecd cells and d cells[1].T

on in clinicaFTIR-basede murine m

on of micrcation of mrentiationofII) [2].

Authors thaEC-2016/22/M

ces Wiercigroch, E

Chem. submithrabaszcz, K. Marzec, S. C

troscopic I: To Detec

grocha,b, Kna M. Mar

mistry, JagiCentre for E

Bobrical Genomi

k we focuseo investigatepurpose we Agilent Tecascular endodothelial celcertain wheectrally obsls derived frextend in th

ments (Fig. ct spectral demonstra

The inflammally applicad methodol

model of brerometastasis

metastatic cefinflamed c

nk the NM/ST4/00150 a

E. Staniszewstted (2017). . Kochan, A.

Chlopicki, Ana

Imaging Uct Inflame

Karolina Crzecb,c, Ma

iellonian UnExperimentarzynskiego ics OMICR

Kr

ed on in vite an effect

employed chnologies aothelial celllls with lungether alternservable andfrom the samhe “spatial”

1 I) and profile of

ated how FTmatory stateable timefralogy was aeast cancer s comparinells was notcells within

National Sand KM:UMO

ska-Slezak, K

Fedorowicz,alyst submitte

Using Staned and Ca

Chrabaszczalgorzata B

niversity, Gral Therapeu14, Krakow

RON, Jagiellrakow, Pola

tro FTIR spof incubatitechnologi

along with als (HMLVEg adenocarc

nation of ind whether ime family b” meaning”

how IR cells. We

TIR spectroe appears aame has an

applied to e(model 4T1

ng to the ht always evilung tissue

cience CenO-2016/23/B/N

K. Szkaradek,

, A. Jasztal, Eed (2017).

ndard andancer Cell

za,b,c, EmilBaranskaa,

ronostajowautics (JCET)w 30-348, Polonian Univand

pectroscopyion with cycal solutiona standard sEC) and celcinoma cellntracellular t gives a co

but with dif inflammatimeasuremealso discu

oscopy imagat an early-n invaluableexaminean e1). Here, wehistologicalident. In ade from matr

Figuinflaspeciinto intradeteclungsmicrcrossand micrinfla

ntre in PNZ4/01379).

T. Wojcik, Y

E. Buczek, L

d High Mls in vitro

lia Stanisz,b, Kamilla

a 2, Krakow), Jagiellonioland versity, Coll

y study on tytokine tumn enhancingset up of thells derived ls (EA.hy92

chemical ommon/difffferent phenion state ca

ents modesussed imageging can be-stage of me diagnosticearly stage e showed a l examinatiddition, we rix of health

ure 1. I. FTIRmed cells: (1) eific for whole nucleus and

cellular compction of micros: (1) H&E ometastasis; (2s-section (grey

(3) high-magometastasis (lmed and cancer

Poland for

Y. Ozaki, M

L.S. Leslie, R

Magnificatiand ex vi

zewska-Slea Maleka,b

w 30-387, Pian Univers

legium Med

two endothemour necros

g spatial ree FTIR micfrom the fu

26). The aimcompositio

ferent spectrnotypes. Finan be determs (transmisse quality oe applied to most diseasec potential.

of lung mrobust FTI

ion for whproposed ahy and can

R-based discrimextraction of sp

cells; (2) diffcytoplasm, an

partments. ometastasis in t

staining with2) IR image o– metastatic ca

gnification IR light blue anr cells, respecti

financial

. Baranska, K

R. Bhargava, K

ion ivo

ezaka,b,

Poland sity,

dicum,

elial cell is factor

esolution roscope.

fusion of m of this on upon ral panel nally, we mined in sion vs.

of single classify

esand its Thus, a

metastasis IR-based hich the a method cer cells

mination of pectral data fferentiation nd (3) into

II. FTIR the murine h labelled

of the lung ancer cells)

image of nd red – vely).

support

K. Malek,

K. Malek,

Latest Advancements in Nanoscale IR Spectroscopy: Spatial Resolution, Speed and Spectral Range

Miriam Unger, Anirban Roy, Eoghan Dillon and Kevin Kjoller

Anasys Instruments, 325 Chapala Street, Santa Barbara, CA 93101, USA

Nanoscale infrared spectroscopy has been successfully demonstrated in an expanding range of applications in recent years due to significant increases in capability. One method of nanoscale infrared spectroscopy, atomic force microscope based infrared spectroscopy (AFM-IR) directly detects IR radiation absorbed by the sample using the AFM probe tip to sense thermal expansion. This thermal expansion depends primarily on the absorption coefficient of the sample and is largely independent of other optical properties of the AFM tip and the sample. One of the initial major improvements in the AFM-IR technique was the development of the resonance enhanced version of AFM-IR. In the resonance enhanced technique, an IR source with a tunable pulsed repetition rate is tuned such that the repetition rate is matched to a contact resonance of the AFM cantilever providing much larger oscillations of the cantilever. This has been employed to investigate samples as thin as an individual monolayer due to an improved sensitivity which is orders of magnitude higher than the non-resonant AFM-IR. Additionally, the resonance enhanced AFM-IR technology has demonstrated 100x faster spectral and 10X faster imaging acquisition times with better SNR. This development is instrumental to augment the reliability of nanoscale characterization by reducing the overall data acquisition time and enabling users to perform repeated measurements for statistical analysis. The resonance enhanced AFM-IR technique has previously been limited to the mid IR range which is accessible with QCL sources. The recent development and integration of a broad range Optical Parametric Oscillator (OPO) source which can be pulsed at rates compatible with resonance enhancement provides an extended spectral range. This has allowed measurements in the C-H, N-H and O-H vibrational stretching range (3600-2700 cm-1) with significantly improved sensitivity. More recently, building on the resonance enhanced AFM-IR technique we have introduced the Tapping AFM-IR mode. This mode allows chemical imaging while in the tapping mode such that soft polymer materials and loose particles can be analyzed. Due to the heterodyne detection method employed in this technique, the spatial resolution of the chemical measurements has improved to better than 10 nm. This presentation will describe the underlying technology including their recent advances and will also highlight numerous biological and life science related applications of nanoscale spectroscopy and chemical imaging. References 1. A. Dazzi, R. Prazeres, F. Glotin, J. M. Ortega, Opt. Lett. 30, 2388-2390 (2005). 2. F. Lu, M. Jin, M. A. Belkin, Opt. Express 29, 19942-19947 (2011). 3. A. Dazzi, C. Prater, Chem. Rev. (2016). DOI: 10.1021/acs.chemrev.6b00448.

Scatterikey techon the 1sharp mat the tithe tip sample.nanoscawith <2Figure charactespecificspectra.reprodudiffers nanoscasecondaalso beeBesidesstructurvalues fdimensi

Referen[1.] F. Ke[2.] C. W[3.] I. Am[4.] R. W

Figure spatial rof a sinin the ab

ExploriU

ng-type scahnologies to10-nm leng

metallic AFMips apex. Thand is mod Detection

ale resolved20 nanomete

1 shows eristiccuticlc absorption Spectra of

ucibility andsignificantl

ale structuraary structureen publisheds non-invasire of biologifor polymerional structu

nces eilmann, R. H

Westermeier, etmenabar, et al.

Wiens, et al., Fa

1.a Topograpresolution at pgle collagen fbsorption ima

ing MicroUsing Infr

anning nearo study the

gth scale – fM tip whichhe light witdified by thof the elas

d near-field er spatial res

a topogrles. nano-FTn lines featuf location 1 d quality ofly for samal composite are obtaind in [4]. ive nanoscaical specimrs or the deures.

Hillenbrand, Pht al., Nature C., Nature Comaraday Discus

phy image ofpositions indicfibril (top). Thage. nano-FTIR

o and Nanrared Nan

M

ne

r-field opticoptical propfar beyond h is externathin the opt

he local diestically tip-s

images [2]solution. raphy scanTIR spectra

uring Amide+ 2 measu

f the spectrmple locatio

tion of the ned for singl

ale resolvedmens, nano-F

etermination

hil. Trans. R. SCommunicatiommunications8ss. 187, 555 (2

f a human hacated in the tohe typical perR spectra of th

nobiologicno-spectro

Max Eisel

easpec Gmb

al microscoperties of phthe diffractlly-illuminatical hotspolectric propscattered lig] and broad

n of the a obtained oe I+II+III bared in the croscopic sigons at the

hair. Simille collagen

d material idFITR enablen of free ca

Soc. Lond. A ns 5, 4102 (20

8, 14402 (20172016).

air (top). nanoopography imaiodic substruche collagen fib

cal Tissue oscopy (Na

le

bH

opy (s-SNOhysical, chetion limit oated to creaot interacts wperties (e.g.ght simultandband near-

surface oon the indicands, in goocentre of thgnatures. Thcuticle rimlar results ofibrils as ca

dentificationes quantitatarrier conce

362, 787 (200014). 7).

o-FTIR spectage. b Topogrcture of the cobril (bottom)

at the Naano-FTIR

OM) has ememical and bf light[1]. s

ate a nanoscwith the sam. absorptionneous to A-field spect

of a humacated positiod agreemee cuticles ohe slope of

m indicatingof nanoscalan be seen in

n or analysive measureentration an

04).

tra (bottom) rraphy and optollagen is resofeature clear A

anoscale R)

merged as onbiological ms-SNOM emcale optical mple surfacn, reflection

AFM imagintra (nano-FT

an hair rions show ment to far-fieoutline the ef the Amideg a changele variationn Fig. 1b w

sis of the seements of dnd mobility

recorded withtical absorptioolved in the AAmid I + II ba

ne of the materials mploys a hot-spot

ce below n) of the ng yields TIR) [3]

revealing material-eld FTIR excellent e I band e in the ns of the

which has

econdary dielectric y in low-

h <20nm on image AFM and ands.

Single-shot Microsecond-resolved Spectroscopy of the Bacteriorhodopsin Photo Cycle with Quantum Cascade Laser Frequency Combs

Markus Mangold1, Jessica L. Klocke2, Pitt Allmendinger1, Andreas Hugi1,

Markus Geiser1, Pierre Jouy3, Jerome Faist3, Tilman Kottke2

1) IRsweep AG, c/o ETHZ, Auguste-Piccard-Hof, Zurich, Switzerland 2) Bielefeld University, Universitaetsstr. 25, Bielefeld, Germany

3) ETH Zurich, Auguste-Piccard-Hof, Zurich, Switzerland

Time-resolved vibrational spectroscopy is an important tool for understanding biological processes as well as chemical reaction pathways [1]. Today, most available methods require many repetitions of an experiment to acquire a microsecond time-resolved mid-infrared spectrum. Therefore, studies of sub-millisecond kinetics are mostly limited to repetitive processes. We present the IRspectrometer, a quantum cascade laser dual frequency comb spectrometer [2-3]. It allows for parallel acquisition of hundreds of mid-infrared wavelengths with microsecond time resolution. The formation of the light-activated L, M and N-states in bacteriorhodopsin – which only have µs to ms lifetimes – has been recorded with the setup shown in Figure 1a). Figure 1b) illustrates the infrared response of bacteriorhodopsin to 10 ns visible light pulses with microsecond time-resolution. The different wavelengths were all measured in parallel thanks to the dual-comb approach. The spectra as well as the kinetics show good agreement with those from step-scan FT-IR measurements. As a benchmark, the spectral signature of several intermediate states of the bacteriorhodopsin photo cycle has been recorded in a single shot measurement. This approach greatly reduces the complexity of time-resolved bio-spectroscopy measurements in the mid-infrared which currently require many repetitions.

Figure 1 a) Setup illustration of the dual-comb quantum cascade laser spectrometer, including a pulsed Nd:YAG laser for activation of bacteriorhodopsin. b) Spectral changes of bacteriorhodopsin with microsecond time resolution at 3 different wavelengths.

[1.] Ritter, Eglof, Ljiljana Puskar, Franz J. Bartl, Emad F. Aziz, Peter Hegemann, and Ulrich Schade. “Time-

Resolved Infrared Spectroscopic Techniques as Applied to Channelrhodopsin.” Frontiers in Molecular Biosciences 2 (July 7, 2015). doi:10.3389/fmolb.2015.00038.

[2.] Hugi, Andreas, Gustavo Villares, Stephane Blaser, H. C. Liu, and Jerome Faist. “Mid-Infrared Frequency Comb Based on a Quantum Cascade Laser.” Nature 492, no. 7428 (December 13, 2012): 229–33. doi:10.1038/nature11620

[3.] Villares, G. et al. Dual-comb spectroscopy based on quantum-cascade-laser frequency combs. Nat. Commun. 5:5192 doi: 10.1038/ncomms6192 (2014).

Mid-IR Photothermal Deflection Spectrometer Based on Quantum Cascade Lasers:

Towards Non-Invasive Glucose Measurement

O. Hertzberg1,2, A. Bauer1,2 and W. Mäntele1

1Institute of Biophysics, Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany

2DiaMonTech GmbH, 10245 Berlin, Germany Photothermal deflection spectroscopy (PTDS) in combination with external cavity tunable quantum cascade lasers (EC-QCL) allows to gather mid-infrared (mid-IR) spectral information from opaque biological samples with a penetration depth comparable to transmission measurements, much higher than for attenuated total reflection IR spectroscopy. To perform in vivo measurements of biological samples such as human skin we propose to use an internal reflection element (IRE) to guide pump and probe beam to the sample interface. In thisconfiguration, the modulated mid-IR pump beam, e.g. from an EC-QCL, irradiates the sample and produces a thermal wave due to absorption that migrates into the IRE. This gradient periodically deflects the path of the visible probe beam, which is monitored by a position-sensitive photodiode detector. This allows to detect mid-IR absorption with cheap vis-optics. [1]Further features of the photothermal technique are the possibility of phase sensitive detection and depth profiling by varying the modulation frequency.This opens multiple applications of mid-IR spectroscopy of complex, multi-layered samples that are difficult to analyze with conventional infrared spectroscopic techniques. [2] We used our laboratory setupto obtain spectral information of human skin in vivoin order to study the properties of the outermost skin layers and to monitor glucose in the interstitial fluid that is closely correlated to blood glucose. [1-3] For further studies, as for example in clinics, we designed a portable and integrated prototype. This prototype will be presented after the talk during the poster session, where we will demonstrate the functionality our spectrometer on various medically relevant test samples.

Concept of total internal reflection enhanced-PTDS [2]

References [1] M. A. Pleitez et al., The Analyst 140, 483 (2015). [2] O. Hertzberg et al., The Analyst 142, 495 (2017). [3] A. Bauer et al., Journal of Biophotonics (2017).

Fun

Depar

MicrobeHowevecomplexdemonsprobingwith consingle microspmicrobemicrofluthe groucells wsubstratcell gencells frmicrofluand horCells nogreen cwhereasare mogreen cmeasurelabeled Raman collectiothe samoutlet). Referen[1] W.E.

Micro[2] S. Ha

C. Ba[3] D. Be

SchmFowle

[4] Y. Wa[5] K.S. L

submi

nctional A

rtment of M

es play an eer, measurinx samples istrate usingg with deutenfocal Ramcell level

pectroscopyes to undeuidic-basedup of Roma

with a distinte) for subnomics (Figrom a samuidic devicrizontal dirot captured ircle) flow s cells captved to the

circle) and ed to determor possessspectrum

on outlet) omple stream

nces Huang, K. Sto

obiol. 9, 1878-ider, M. Wagn

aranyi, E.R. Toerry, E. Mader

mid, B.T. Hanser, W.E. Huanang, W.E. HuLee, M. Palatiitted

Analyses a

MicrobiologyMicro

essential rolng the actincluding th

g chlamydiaerated water

man microsp[1, 2, 3,

y for quantierstand thed Raman actn Stocker (Enct Raman

bsequent cugure 1). In

mple fluid ce and focurections by

by the optdirectly int

tured by the sample-fr

have theirmine wheth another de(and thus

or not (and tm and relea

oecker, R. Gri-89 (2007). ner, M.C. Schoenshoff, J. Mr, TK Lee, D Wson, N. Shterzeng, M. Wagneuang, L. Cui, Minszky, J. Ngu

and TargeRaman M

Mic

y and Ecosyobiology, U

le for all ecivity and chheir eukaryoae and gutr and/or 13C

pectroscopy 4]. Furthe

ifying the seir role in tivated cell ETH Zürich

n spectrum ultivation on the RACS

are flownused in the

two sheathtical tweezeto the wasthe optical tree stream r Raman s

her they are esired peak

released thus carried

ased into th

iffiths, L. New

hmid, B.S. SixMontanaro, M.Woebken, Y. Wer, I. Mizrahi,

er, Proc Natl AM. Wagner, Cuuyen, V. I. Fer

eted SinglMicrospec

chael Wag

ystem Scien

University of

cosystems aharacterizinotic hosts is t microbiotC-labeled co

to characteermore, I storage com

wastewatesorter (RAC

h) for high-t(demonstr

or single S device n into a

vertical h flows.

ers (solid te outlet, tweezers (dashed

spectrum isotope-

k in their into the

d back to he waste

wbold, H. Dai

xt, J.G. Christi Horn, Mol MWang, D. Zhu, I. Rauch, T. D

Acad Sci U S Aurr Opin Biotrnandez, F. M

le Cell Genctroscopy

gner

nce, Researcf Vienna, A

and for the hng the func

extremely ta as examompounds cerize in vivowill demon

mpounds ofer treatmenCS) that wethroughput ating e.g.

ms, A.S. Whi

ian, G. HäckerMicrobiol. 77, u, M. PalatinsDecker, T. BoA. 112(2), E19echnol. 41, 34enolascina, FC

Figure 1.Deof the Ramansorting (RAC

nomics of

ch NetworkAustria

health of antion of micchallenging

mple show can be appl

o the functionstrate howf biotechnont. Finally, e have devesorting of iuptake of

iteley, M. Wag

r, P. Pichler, K687-700 (201zky, A. Schinocklitz, J. Pop94-203 (20154-42 (2016). C. Pereira, D.

sign and workn-activated mCS) system [5

f Microbes

k Chemistry

nimals and crobial cellg. In this talsingle cell lied in comon of microw we use

ologically imI will pr

eloped togetndividual man isotope

gner, Environ

K. Mechtler, A0).

ntlmeister, M.Cp, C.M. Gibso).

Berry, M. W

king principlemicrobial cell

]

s by

meets

humans. s within lk, I will

isotope mbination obes on a

Raman mportant resent a ther with microbial e-labeled

A. Müller,

C. on, P.W.

agner

Developing Spectroscopic Processes for the Detection of Bacteria: Environmental, Surface Deposited and High Throughput

Claire L. Pickering1,3, Angela M. Flack1,2, Ganesh D. Sockalingum2,

Roy Goodacre3, Matthew J. Baker1*

1WestCHEM, Department of Pure and Applied Chemistry, Technology and Innovation Centre, 99 George St, Glasgow, G1 1RD, UK

2Equipe MeDIAN-Biophotonique et Technologies pour la Sante, Universite de Reims Champagne-Ardenne, CNRS UMR 7369-MEDyC, UFR de Pharmacie, 51 rue Cognacq-Jay,

51096 Reims Cedex, France 3Manchester Institute of Biotechnology, Department of Chemistry, University of Manchester,

131 Princess Street, M1 7DN, UK

*matthew.baker@strath.ac.uk @ChemistryBaker

Abstract Biological weapons represent an invisible threat and could prove to be devastatingly effective. Defence against the use of bacterial biological warfare agents (BWAs) is becoming an increasingly important concern, which is reflected in the National Security Strategies of the USA and UK. The UK has highlighted international terrorism affecting the UK or its interests, including a chemical biological, radiological or nuclear attack by terrorists as a tier one risk1. The USA specifically mentions countering the biological threat to strengthen resilience across the spectrum of high-consequence biological threats2. This paper will first of all discuss the use of spectroscopy combined with pattern recognition algorithms and its use for detecting surface deposited BW simulants and the impact of environmental conditioning of these simulants on the spectroscopic signatures and pattern recognition models. The temperature and humidity conditions used are within the ranges prescribed in the Ministry of Defence Standard on Natural Environments and measurements from Camp Bastion, Afghanistan. The mode of spectroscopic analysis is important with a plethora of tools required for different situations. For instance, sampling requirements required on the front line may differ than those required for analysis in a tertiary well-equipped microbiological identification laboratory. However, the central tenants of reduced cost, ease of sample preparation, rapid acquisition and data analysis speed to enable economic savings and reduced time to result. This paper will discuss and highlight possible uses, advantages and disadvantages of these approaches. References [1] A Strong Britain in an Age of Uncertainty: The National Security Strategy, CM7953, October 2010. [2] National Security Strategy 2010, United States Government, May 2010.

Direct Characterization of Stored Red Blood Cells Using Raman Spectroscopy

Rekha Gautam1, Joo-Yeun Oh2, Rakesh Patel2, Richard A. Dluhy1*

1Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294 USA

2Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294 USA

Donated red blood cells (RBCs) continue to metabolize via the glycolytic pathway and degrade during storage in blood bags at 4-6°C. Transfusion with older, degraded blood products underlies poorer therapeutic efficacy, and the potential fortransfusion-associated toxicity. Current methods used for RBC screening are invasive and time-consuming. Thus, improving diagnostic ability to assess the quality in a given until of stored RBC remains a key goal in transfusion medicine. Resonance Raman spectroscopy is a label-free modality that provides enhanced Raman spectra of molecules associated with electronic transitions in resonance with the excitation wavelength. In this study, we discuss recent efforts in developing a novel strategy - Diffuse Resonance Raman Spectroscopy (DRRS) – to acquire depth-sensitive Raman spectra. Two main aspects of this methodology include i) illumination by a diffuse excitation beam which decreases photon density and penetrates further into the sample, and ii) inclusion of additional CCD detector pixels adjacent to those aligned with the optical path to augment the detection of diffuse photons. Unlike excitation with near-infrared wavelengths, DRRS requires ten-fold less power and acquisition time to get a comparable signal-to-noise ratio. We employed this approach for identification of the spectral features of hemoglobin that characterized the age-dependence of RBCscontained inside blood bag segments underneatha 1 mm polymer layer. Statistical analysis performed on these Raman spectra classified young (6-8 days) and old (35-42 days) stored RBCs with >95% sensitivity and specificity. Based on these results, it is evident that DRRS is a rapid, non-invasive and non-destructive approach to attain subsurface information for RBC-related molecules in resonance with the excitation wavelength, and has the potential for in-vivo applications targeting molecules such as hemeproteins and carotenoids.

Application of FT-IR and MALDI-TOF MS for Bacterial Typing: A Current Standpoint

Luísa Peixe

REQUIMTE@UCIBIO. Faculty of Pharmacy, University of Porto, Porto, Portugal

Precise and quick identification of specific bacterial taxonomic units can have

a tremendous impact in the management of clinical or food-related outbreaks and epidemiological surveillance of pathogenic and/or antibiotic resistant strains, being the development of reliable, quick and low cost alternatives one of the priority areas of research assumed by several world health organizations.

In recent years, whole genome sequencing (WGS) demonstrated to be useful for bacterial typing, and especially for large outbreak management, at a more affordable cost. Alternative typing methods based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Fourier-Transform Infrared Spectroscopy (FT-IR) appeared to have attractive turnaround times and sufficient resolution power to assist epidemiological surveillance and outbreak control. This presentation will cover our expertise in the application of these methodologies for the discrimination of bacterial species (Acinetobacter baumannii complex), serotypes (Salmonella enterica) and clones of different clinically relevant species (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium), and their advantages/drawbacks in comparison with competitor technologies/approaches.

Primary Cells vs. Cell Lines: in vitro Experiments and Spectroscopic Analysis

E. Szafraniec1, S. Tott1, A. Rygula1, K. Malek1,2, M. Baranska1,2

1 Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, Krakow, Poland

2Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow, Poland

For decades, cell lines have played a critical role in scientific advancements, as they are readily available and allow for a more controlled manipulation of cellular functions and processes. Therefore, cell culture studies provide a valuable complement to in vivo experiments, where highly complex situation takes place. In contrast to cell lines, primary cells which are isolated directly from tissues, have a finite lifespan and limited expansion capacity. On the other hand, primary cells have normal cell morphology and maintain many of the important markers and functions seen in vivo.[1-3] Therefore, in many cases the use of cell line is desirable e.g. cell biological studies on basic mechanism or in drug development, however, in other cases such as research on complex metabolic processes, the use of primary cells is more suitable.[4] When the choice is not obvious, it is worth to complement the results obtained from cell culture using the primary cells. Over the years Raman spectroscopy imaging becomes well established technique that enablesstudding subcellular processes, due to its submicron resolution and sensitivity and specificity. Great part of such studies is based on cell culture experiments, however the use of primary cells becomes more and more desirable. Spectroscopic imaging provides not only spatial information, possible to achieve with other microscopic techniques, but also the specific, spatially localized knowledge about chemical composition of the sample. Here we utilized this technique to establish the diversity between immortalized cell line derived from mouse cell lines and freshly isolated primary cells. The results are presented for LSECs (Liver Sinusoidal Endothelial Cells) and CMECs (Cardiac Microvascular Endothelial Cells) isolated from the liver tissue and heart, respectively. Moreover, data for eosinophils isolated from blood and respective cell line is also discussed. This work was supported by National Science Centre (NCN, grant No. UMO-2015/16/W/NZ4/00070 and UMO-2016/22/M/ST4/00150). References [1] G. Kaur and J. M. Dufour, Spermatogenesis 2, 1–5 (2012). [2] D. Bouis, G. A. P. Hospers, C. Meijer, G. Molema and N. H. Mulder, Angiogenesis 4, 91–102 (2001). [3] C. S. Alge, S. M. Hauck, S. G. Priglinger, A. Kampik and M. Ueffing, J. Proteome Res. 5, 862–878 (2006). [4] C. Pan, C. Kumar, S. Bohl, U. Klingmueller and M. Mann, Mol. Cell. Proteomics 8, 443–450 (2009).

Cross-border Salmonellosis Outbreak Linked to Fresh Sprouts – Analysis of Salmonella Bovismorbificans - Isolates by FTIR-Spectroscopy –

Helene Oberreuter1, Elisabeth Aichinger2, Maja Adam2, Jörg Rau1

1Chemical and Veterinary Investigations Office (CVUA) Stuttgart, Germany

Schaflandstr. 3/2, D-70736 Fellbach Helene.Oberreuter@cvuas.bwl.de 2Baden-Württemberg State Health Office (LGA), Germany

Nordbahnhofstr. 135, D-70191 Stuttgart  In July of 2014, southern German surveillance authorities noted a salmonellosis outbreak in-volving>60 human cases, followed by additional >20 cases in Switzerland in subsequent weeks. As a combined causative agent, Salmonella enterica ssp. enteric serovar Bovismorbi-ficans [6,8:r:1,5] was identified. Through cooperation between local health authorities, the Baden-Württemberg State Health Office (LGA) and food surveillance authorities, contami-nated fresh sprouts were assumed to be the source of the outbreak [1, 2]. Distributed by a wholesaler, the sprouts had been used for decoration of fresh salads mainly in the German state districts of Konstanz and Friedrichshafen.

The initial isolation of salmonella isolates from the contaminated foods was done at the CVUA Stuttgart. Sprout samples were analyzed in line with the official collection of analysis methods according to §64 of the German Food and Feed Code (LFGB), followed by subse-quent serotyping. Genus confirmation was achieved by MALDI TOF mass spectrometry. An outbreak patient’s isolate, provided by the LGA, was included in the analyses. All outbreak isolates were evaluated by FTIR spectroscopy within the setting of other salmonella strains including those of the same serovar. All outbreak sample spectra were correctly allocated to O-serogroup C2-C3 (O:8) by means of a previously established Artificial Neural Network for differentiation of salmonella O-serogroups by FTIR spectroscopy [3].

Due to the spectroscopical alignment between food and human isolates on the basis of the consumption anamneses of patients, the initial assumption of sprouts as being the outbreak’s source was confirmed. As previously demonstrated (i.a. [4]), FTIR spectroscopy again proved a convenient tool for a quick alignment of different isolates in the case of a food-related out-break. References [1] C. Wagner-Wiening, D. Lohr, A. Diedler, Infektionsbericht Baden-Württemberg 30, 1 (2014). [2] C. Wagner-Wiening, D. Lohr, A. Diedler, Infektionsbericht Baden-Württemberg 34, 3-4 (2014). [3] H. Oberreuter, J. Rau, Dt Lebensmittel-Rundschau 111(12), 498-502 (2015). [4] A. Fetsch et al., Int J Food Microbiol 187, 1-6 (2014).  

  1

Advanced IR Nanospectroscopy to Study Lipids Bodies in Micro-organisms: Toward a Better Understanding of Metabolic Pathways at Stake

A. Deniset-Besseau1, R. Rebois1, J.M. Nicaud2, M.J. Virolle3, O. Goncalves4, A. Dazzi1

1Laboratoire de chimie Physique, Université Paris-Sud, 91405 Orsay –France 2Micalis Institute, INRA, AgroParisTech, Jouy-en-Josas, France

3 I2BC, Université Paris-Sud , Orsay, France 4 GEPEA, Saint-Nazaire, France

Abstract Infrared spectroscopy is a powerful tool to specifically probe molecular vibrations of organic components without exogenous labelling and becomes even appealing to cellular and tissue biology. The coupling with microscopy and all the improvements done in the last decade have reinforced this trend. The main drawback is still remaining: the resolution limited by the diffraction. The solution to the problem was in the side of the near-field techniques. After several years of research in the field and considering all the previous methods limitations, we have developed at our lab an innovative infrared nanospectromicroscopy : AFMIR (Alexandre DAZZI, patent 2007)1,2. AFMIR is a cutting-edge near-field technique coupling an atomic force microscope (AFM) with a tunable pulsed IR laser. This allows IR mapping of molecules of interest within a sample with a sensitivity of ten of nanometer. We will present the study of lipid bodies accumulated in the different microorganisms (bacteria, microalgae and yeast). Those lipid accumulations consist mostly in triacylglycerols (TAG) and steryl esters. It was possible thanks to our AFM-IR system to create sub-cellular chemical maps that allows label-free identification of TAGs inclusions in the cytoplasm. To carry out these studies new set-up configurations were required as well as the use of correlative imaging. We will also discuss the capability of AFM-IR technique to provide new insights into the constitution of the fatty inclusions and the role of TAGs in the morphological and metabolic differentiation that characterize those micro-organisms. References [1] A. Dazzi, F. Glotin, R. Carminati, Theory of Infrared Nanospectroscopy by Photothermal Induced

Resonance, J. Appl. Phys. 107, 1–7 (2010). [2] B. Lahiri, G. Holland, A. Centrone, Chemical Imaging beyond the Diffraction Limit: Experimental

Validation of the PTIR Technique, Small 9, 439–45 (2013). [3] A. Deniset-Besseau, C. Prater, M.-J. Virolle, and A. Dazzi, J. Phys. Chem. Lett. 5 (4), 654–658 (2014).

Non-Contact Methodology for Obtaining Submicron IR Spectra and Images of Cells and Tissue

Curtis Marcott1, Mugdha Padalkar2, Jessica M. Falcon2, Nancy Pleshko2

1Light Light Solutions, Athens, GA, USA

2Department of Bioengineering, Temple University, Philadelphia, PA, USA

Nanoscale infrared spectroscopy has been successfully demonstrated in an expanding range of applications in recent years due to significant increases in capability. One method of nanoscale infrared spectroscopy, atomic force microscope based infrared spectroscopy (AFM-IR) directly detects IR radiation absorbed by the sample using the AFM probe tip to sense thermal expansion. This thermal expansion depends primarily on the absorption coefficient of the sample and is largely independent of other optical properties of the AFM tip and the sample. The use of a quantum cascade laser (QCL) as the excitation source has dramatically improved the sensitivity of AFM-IR and enabled spectra and absorbance images to be collected much more rapidly. A breakthrough new optical technique capable of producing submicron IR spectra and images will be described. Example applications of this new submicron non-contact methodology to cells and tissues will be presented.

Cultivation-free Identification of Lung Bacteria by Means of Raman Spectroscopy

P. Rösch1,2, B. Lorenz1,2, C. Wichmann2,3, S. Stöckel1,2, S. Meisel1,2, A. Silge1,2,

T. Bocklitz1,2,3, J. Popp1,2,3

Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany.

InfectoGnostics Research Campus Jena, Center for Applied Research, Philosophenweg 7, D-07743 Jena, Germany

Leibniz Institute of Photonic Technology e.V., Albert-Einstein-Straße 9, D-07745 Jena, Germany

The identification of lung pathogens is sometimes time consuming since a couple of bacteria require cultivation for several days or even weeks like e.g. Legionella spp. or Mycobacteria spp. Applying Raman microspectroscopy in combination with destruction-free isolation techniques allows for a fast identification of bacteria without the need for cultivation [1-3]. Since Raman spectroscopy is a phenotypic method, changes in the biochemical composition of the cells will show up in the spectral information. Therefore, monitoring of the physiological state of the bacteria is necessary [4]. Especially, Raman spectra of the same species differ significantly according to their state as planktonic and sessile cells [5] or as intracellular bacteria [6, 7]. Taking such premises into account, even species of the huge and diverse genusMycobacteria can successfully be identified by means of Raman microspectroscopy [8]. Acknowledgment Funding of the research projects InterSept (13N13852) and InfectoGnostics (13GW0096F) from the Federal Ministry of Education and Research, Germany (BMBF) as well as the Leibniz project “Lung Microbiota” (SAW-2016-FZB-2) is gratefully acknowledged. References [1] S. Pahlow, S. Meisel, D. Cialla-May, K. Weber, P. Rösch and J. Popp, Adv. Drug Deliv. Rev. 89, 105-120

(2015). [2] S. Stöckel, J. Kirchhoff, U. Neugebauer, P. Rösch and J. Popp, J. Raman Spectrosc. 47, 89-109 (2016). [3] B. Lorenz, C. Wichmann, S. Stöckel, P. Rösch and J. Popp, Trends Microbiol. 25, 413-424 (2017). [4] S. Stöckel, A. S. Stanca, J. Helbig, P. Rösch and J. Popp, Anal. Bioanal. Chem. 407, 8919–8923 (2015). [5] D. Kusić, B. Kampe, A. Ramoji, U. Neugebauer, P. Rösch and J. Popp, Anal. Bioanal. Chem. 407, 6803–

6813 (2015). [6] D. Kusić, A. Ramoji, U. Neugebauer, P. Rösch and J. Popp, Anal. Chem. 88, 2533-2537 (2016). [7] A. Silge, E. Abdou, K. Schneider, S. Meisel, T. Bocklitz, H.-W. Lu-Walther, R. Heintzmann, P. Rösch and

J. Popp, CellMicrobiol. 17, 832–842 (2015). [8] S. Stöckel, S. Meisel, B. Lorenz, S. Kloß, S. Henk, S. Dees, E. Richter, S. Andres, M. Merker, I. Labugger,

P. Rösch and J. Popp, J. Biophotonics 10, 727-734 (2017).

Model-based Pre-processing for Estimating Scattering and Absorption in Infrared Spectroscopy

Achim Kohler1,2, Johanne Solheim1, Tatiana Konevskikh2, Maren Anna

Brandsrud1, Harald Martens3,4, Carol Hirshmugl5, Reinhold Blümel6

1Faculty of Science and Technology, Norwegian University of Life Sciences Drøbakveien 31, 1430 Ås, Norway

2MWT Analytics AS, 1430 Ås, Norway 3Idletechs AS, 7034 Trondheim, Norway4Department of Engineering Cybernetics, Norwegian

U. of Science and Technology, 7034 Trondheim, Norway, 5University of Wisconsin-Milwaukee, Milwaukee, WI. 532010340, USA

6Department of Physics, Wesleyan University, Middletown, Connecticut 06459-0155, USA Strong scattering effects appear when the wavelength of the electromagnetic radiation used in a spectroscopic method matches the size of the measured sample. Examples are Mie scattering and fringes. In infrared spectroscopy of cells and tissues Mie-type scattering occurs[1]. Cells and cell components have an approximate spherical shapeand cause strong Mie-type scattering effects.When parallel surfaces of materials are involved, so-called fringes are strongly distorting infrared spectra [2]. Scattering effects have been considered as a major obstacle for the interpretation and further use of spectra from infrared microscopy.In addition to scattering effects, other unwanted effects such as water and water vapor variations appear in the infrared spectroscopy of biological materials. In collaboration with several groups, we have during recent years developed algorithms for separating and correcting Mie-type scattering in infrared microspectroscopy [3-6], based on extensions of the Extended Multiplicative Signal Correction. In this paper,we present the latest developments of these algorithms.We discuss the diversity of scattering signals related to Mie-type scattering. We further present an algorithm based on optimization [7] that can be used for estimating and removing unwanted variation patterns such as variations due to water or water vapor in infrared spectra. References [1] B. Mohlenhoff, M. Romeo, B.R. Wood, M. Diem, Biophys. J. 88, 3635–3640 (2005). [2] T. Konevskikh, A. Ponossov, R. Blumel, R. Lukacs, A. Kohler, Analyst 140, 3969-3980

(2015). [3] P. Bassan, A. Kohler, H. Martens, J. Lee, H.J. Byrne, P. Dumas, E. Gazi, M. Brown,

N. Clarke, P. Gardner, Analyst 135, 268-277 (2010). [4] T. Konevskikh, R. Lukacs, A. Kohler, Journal of Biophotonics (2017). [5] T. Konevskikh, R. Lukacs, R. Blumel, A. Ponossov, A. Kohler, Faraday Discuss. 187,

235-257 (2016). [6] A. Kohler, J. Sule-Suso, G.D. Sockalingum, M. Tobin, F. Bahrami, Y. Yang, J. Pijanka,

P. Dumas, M. Cotte, D.G. van Pittius, G. Parkes, H. Martens, Appl Spectrosc 62, 259-266 (2008).

[7] H. Martens, Chemometrics and Intelligent Laboratory Systems 107, 124–138 (2011).

Figure 1.Burkholderia contaminans LMG 23361 biofilms grown in modified M9 minimal medium on a CaF2 slide (d = 0.5 mm) for 48 hours. The air-dried biofilm was studied using AFM (topography images of panels A, B), CRM (cf. panel C) and other methods. The corresponding mean Raman spectra are given by panel D.

A

DB

2935

1670

1455

1320

1250

1050

1007

100015002000250030003500

Ram

an In

tens

ity

1735

2935

Wavenumber cm-1

C1

2 3

1

23

1

2

3

Vibrational Micro- and Nanospectroscopy of Burkholderia contaminans Biofilms

Cecilia Figoli1, Maren Stämmler2, Beltina León1, Christoph Schaudinn3,

Alejandra Bosch1, Michael Laue3, Peter Lasch2

1 CINDEFI, CONICET-CCT La Plata, Facultad de Ciencias Exactas, UNLP, Argentina; 2 Proteomics and Spectroscopy (ZBS 6), and 3 Advanced Light and Electron Microscopy

(ZBS 4), Robert Koch-Institute, 13353 Berlin, Nordufer 20, Germany

Among the Burkholderia cepacia complex species, B. contaminans represents the most frequently recovered from patients with cystic fibrosis (CF) in Argentina, and its incidence is currently increasing in Portugal and Spain [1]. Although B. contaminans respiratory tract infection may be transient, its acquisition most frequently results in a chronic infection with unfavorable clinical manifestation anda gradual decline in lung function [2]. The adaptation strategies leading to the establishment of these bacteria in CF lungs are still unknown; nevertheless biofilm formation may be playing a role in protecting bacteria from antimicrobial treatments and host immune system. A biofilm is characterized by aggregated cells surrounded by a self-produced matrix which contain polysaccharides, and other components such as eDNA, proteins, and lipopolysaccharides [3].

In this work we present data obtained from Burkholderia contaminans LMG 23361 biofilms produced under batch conditions on CaF2 windows. A tandemimaging analysis by optical light microscopy, atomic force microscopy (AFM), FTIR hyperspectral imaging, confocal Raman microspectroscopy (CRM), confocal laser scan microscopy (CLSM) and scanning electron microscopy (SEM) was developed. The combined application of these techniques allowed studying the physical and chemical heterogeneity of bacterial biofilms at the micro- and nanoscale levels. This novel correlative approach enabled us to get insights into the spatial phenotypic heterogeneity of biofilms, to analyze the individual cell phenotypes and to characterize the biofilm's matrix composition with very high spatial resolution. References [1] P. Martina, M. Bettiol, C. Vescina, P. Montanaro, M. Mannino, C. Prieto, C. Vay, D. Naumann, J. Schmitt,

O. Yantorno, A. Lagares, A. Bosch, J Clin Microbiol 51, 339–344 (2013). [2] P. Martina, S. Feliziani, C. Juan, M. Bettiol, B. Gatti, O. Yantorno, A. Smania, A. Oliver, A. Bosch, Int J

Med Microbiol 304, 1182–1191 (2014). [3] N. Høiby, T. Bjarnsholt, C. Moser, P. Jensen, M. Kolpen, T. Qvist, K. Aanæs, T. Pressler, M. Skov, O.

CiofuAPMIS 125, 339–343 (2017).

Applicability of SERS in Combination with Stable Isotope Approach for Characterization of Microorganisms at Single Cell Level

R. Weiss, P. Kubryk, M. Seidel, R. Niessner, M. Elsner, N. P. Ivleva

Institute of Hydrochemistry and Chair of Analytical Chemistry and Water Chemistry,

Technical University of Munich, Marchioninistr. 17, 81377 Munich, Germany

Microorganisms play a vital role in most ecosystems and for eukaryotes, including humans. They are essential for global biogeochemical cycles as well as for health and disease(s) in the human body. Therefore, it is crucial to developreliable, accurate and sensitive methods for the detection, discrimination andidentification of microorganisms and for the analysis of their interactions with the (a) biotic environment [1-3]. Raman Microspectroscopy (RM) in combination with a stable isotope approach is an emerging tool for the nondestructive characterization of the molecular and isotopic composition of microorganisms at the single cell level, which allows for in situ investigationsof ecophysiology and metabolic functions of microbial communities. When atoms in a molecule are (partially) replaced by heavier stable isotopes, the corresponding Raman bands show a characteristic shift toward lower frequencies (so called red-shift) in the spectrum. Therefore, RM has a great potential for the analysis of stable-isotope tracer incorporation into biomass[1-4].However, disadvantage of RM is a limited sensitivity due to low quantum efficiency of the Raman effect (typically 10-8 – 10-6).

We explored the applicability of Surface-Enhanced Raman Scattering (SERS) in combination with stable isotope labeling for the characterization of single cells. Bacteria of E. coli were grown with substrates (fully) labeled with the stable isotopes 13C, 15N or 2H or on compounds with natural abundance of isotopes. The use of Ag nanoparticlessynthesized in situallowed us to achieve reproducible SERS spectra [5]. The spectra of bacteria are usually characterized by a pronounced band at around 730 cm-1, which was assigned to glycosidic ring vibrations or to adenine in different studies. For 13C- and 15N-labeled cells we found a reproducible red-shift of this band from 733 cm-1 to 720 cm-1 (13C), 717 cm-1 (15N) or even 707 cm-1 (13C&15N) for stable isotope-labeled bacteria. This allowed us to assign this SERS band to adenine-related compounds [6]. Recently Premasiri et al. [7], confirmed that SERS spectra are dominated by contributions of free purine bases: adenine, hypoxanthine,xanthine, guanine, uric acid, and AMP.Furthermore, it has been shown that in contrast to living bacterial cells, no SERS signals can be detected from dead cells [8]. However, our detailed investigations of different Gram negative and Gram positive bacteria revealed that, despite the successful deposition of Ag nanoparticles on the bacterial surface, also living bacteria often exhibit no or (very) weak SERS signal(s). Moreover, the number of hits and the signal intensity by SERS analysis strongly depend on the sample storage time, but no clear correlation with the amount of the living cells (detected by Flow Cytometry and Colilert test) were observed. Thus, we assume that the SERS signal reflects the metabolic activity of bacterial cells. Our findings can open new possibilities for the application of SERS (in combination with a stable isotope approach) to probe for the metabolic activity of microorganisms at the single cell level. References [1.] D. Berry, E. Mader, T. K. Lee et al., Proc Natl Acad Sci USA. 112, E194-E203 (2015). [2.] Y. Wang, W. E. Huang, L. Cui, M. Wagner, Curr Opin Biotechnol. 41, 34-42 (2016). [3.] H. Muhamadali, A. Subaihi, M. Mohammadtaheri et al., Analyst. 141, 5127-5136 (2016). [4.] N. P. Ivleva, P. Kubryk, R. Niessner, Anal Bioanal Chem. 409, 4353-4375 (2017). [5.] P. Kubryk, J. S. Kölschbach, S. Marozava et al., Anal Chem. 87, 6622-6630 (2015). [6.] P. Kubryk, R. Niessner, N. P. Ivleva, Analyst 141, 2874-2878 (2016). [7.] W. R. Premasiri, J. C. Lee, A. Sauer-Budge et al., Anal Bioanal Chem. 408, 4631-4647 (2016). [8.] H. Zhou, D. Yang, N. P. Ivleva, et al., Anal Chem. 87, 6553-6561 (2015).

FT-IR Applied to Microbial Identification and Stress Response Quantification: A Preliminary Balance and New Perspectives

Gianluigi Cardinali1,2, Laura Corte1, Luca Roscini1, Debora Casagrande

Pierantoni1, Daniele Fioretto2,3, Vincent Robert4, Mariangela Cestelli Guidi5, Giuseppe Bellisola6

1 Department of Pharmaceutical Sciences, University of Perugia – Italy,

2 CEMIN Excellence Research Center – University of Perugia, 3 Department of Physics and Geology- University of Perugia – Italy,

4Westerdijk Institute of Fugal diversity – Utrecht –NL, Biomedical Research Center, 5INFN - LNF, SINBAD Lab -Italy,

6associated to INFN - LNF, SINBAD Lab – Italy The use of IR spectroscopy was proposedfor microbial characterization, with the perspective of developing and effective identification tool (1). In spite of the wealth of papers demonstrating the feasibility of FTIR based microbial identification in both, bacteria and fungi (2, 3), this methodology has not yet been widely accepted as a gold standard by microbiologists. FT-IR identification has the advantage of giving a low consumable cost, fast and relatively easy method applicable to almost all microorganisms, although with some technical difficulties for filamentous fungi. On the other hand, the method suffers of the lack of large shared free libraries based on a large number of well identified and taxonomically significant strains. In fact, it has been demonstrated with the use of DNA molecular markers that the yeast identification at the species level requires a deep knowledge of the species structure and of the distribution of the admissible threshold of similarity. Moreover, stringent criteria for the determination of the distance between the unknown strain and the type strains of the most related candidate species are mandatory to make every identification method taxonomically sound (4). These taxonomic aspects must be added to the various aspects related to the reproducibility and reliability of the spectroscopic analyses, contributing to produce a complex situation, calling for future shared actions aiming at transforming this very important and promising technique in a largely used gold standard technique. These considerations can be transferred to the RAMAN technology with some technical differences, but keeping the same conceptual and scientific mainframe. FT-IR analysis was also demonstrated to be an excellent tool to determine the physiological status of the cells of various species and hence to quantitatively evaluate the metabolomics effects of chemical or physical stresses (5, 6).mi This contribution aims at analyzing the actual possibilities of FT-IR based microbial identification, its limits and potential developments and to revise the state of the art of FT-IR evaluation of the microbial cell stress.

References [1] D. Naumann, D. Helm and H. Labischinski, Nature 351, 81-82 (1991). [2] P. Zarnowiec, L. Lechowicz, G. Czerwonka and W. Kaca, Current medicinal chemistry 22, 1710-1718 (2015). [3] A. Lecellier, J. Mounier, V. Gaydou, L. Castrec, G. Barbier, W. Ablain… and G. Sockalingum, Int. J. Food

Microbiology 168, 32-41 (2014). [4] D. Vu, M. Groenwald, S. Szocke, G. Cardinali … and V. Robert, Studies in mycology 85, 91-, 105 (2016).

[5] A. Perromat, A.M. Melin, C. Lorin and G. Deleris, Biopolymers 72, 207-216 (2003).

[6] L. Corte, P. Rellini, L. Roscini, F. Fatichenti and G. Cardinali, Analytica Chimica Acta 659, 258-265 (2010).

Advancing Mid Infrared Imaging Technology: The Mid-TECH Project

M. Hermes1, J. Nallala1, L. Huot2, S. Junaid2, Y. Matsuoka3, J. Tomko3, P. Tidemand-Lichtenberg2, C. Pedersen2, W. T. Masselink3 and N. Stone1

1Biomedical Physics, School of Physics and Astronomy, University of Exeter, EX4 4QL, UK

2DTU Fotonik, Frederiksborgvej 399, 4000 Roskilde, Denmark 3Humboldt University, Newtonstr. 15, D-12489 Berlin

The use of IR transmission spectral profiles for diagnostic purposes has been shown in several proof of concept studies; for example discriminating tissue pathology for diagnosis of adenocarcinoma in the oesophagus [1]. Despite the fact of their usefulness, IR spectroscopic techniques are still not fast to be used in daily clinical routines. Imaging a whole tissue section from an oesophagus biopsy with conventional FT-IR microscope, even with an imaging detector, can take up to 14 hours. For the use in a clinical environment measurement times need to be decreased into the order of minutes to enable large studies and useful real-time spectral pathology tools. A promising solution for this problem could be the use of more efficient detection systems and higher brightness IR light sources, such as quantum cascade lasers (QCL) or supercontinuum sources. Our work in the Marie Skłodowska-Curie Innovative Training Network Mid-TECH evaluates those light source technologies in combination with a new detection scheme called upconversion [2]. The signal in the IR is mixed with a NIR wavelength laser to achieve sum frequency generation and detect in the near visible wavelength range. This allows the use of silicon based CCDs which have a much better noise-equivalent power than conventional IR detectors while also being significantly cheaper. This talk will give an overview on these new technologies and compare advantages and drawbacks of their use benchmarked against the state of the art.

Fig. 1Upconversion image of a tissue section, from a biopsy obtained from a patient with an adenocarcinoma in the oesophagus,  illuminated with  a  QCLat  6µm  1666  cm‐1.  The  upconversion  approach  here mixed  the  IR  light with 1064nm in anAGS non‐linear crystal. 

  The data presented in figure 1 was obtained using QCL upconversion setup. Data obtained from this setup will be compared to work using a supercontinuum setup [3] as well as a conventional Globar-FTIR setup. A potential outline for their use in a clinical environment will be discussed. References [1] O. J. Old, G. R. Lloyd, J. Nallala, M. Isabelle, L. M. Almond, N. A. Shepherd, C. A. Kendall, A. C. Shore, H. Barr and N. Stone,

“Rapid infrared mapping for highly accurate automated histology in Barrett's oesophagus”, Analyst, In Press.(2017). [2] J. S. Dam, C. Pedersen and P. Tidemand-Lichtenberg, “High-resolution two-dimensional image upconversion of incoherent light“,

Opt. Lett. 35, 3796-3798 (2010). [3] L. Huot, P. M. Moselund, P. Tidemand-Lichtenberg, L. Leick and C. Pedersen, “Upconversion imaging using an all-fiber

supercontinuum source“, Opt. Lett. 41, 2466-2469, (2016).

Results from a Large-scale Lung Cancer Spectral Histopathology (SHP) Study

Max Diem

Dept. of Chem. & Chem. Biol. Northeastern University, Boston, MA 02115 (emeritus) and

CIRECA, LLC, Cambridge, MA 02139

In 2015, a 328-patient lung cancer study in tissue micro-array (TMA) format was started at CIRECA, LLC in collaboration with a National Cancer Center in California, City of Hope (COH) Medical Center. The major goals of this study were the discrimination of normal from cancerous tissue regions, the distinction between adenocarcinomas (ADCs) and squamous cell carcinomas (SqCCs), collectively referred to as non-small cell lung cancer (NSLC), and further sub-typing of ADCs. The methodology employed for data acquisition, annotation and classifier training have been detailed in the literature [1].

The balanced accuracy (defined as the mean of sensitivity and specificity) achieved for the first goal, the classification of normal vs. non-small-cell lung cancer, was 97%. The accu-racy of the second goal, the clinically and therapeutically relevant distinction of ADC from SqCC was 92 %. The sub-classification of ADC revealed interesting insights into the correla-tion between SHP results and therapeutic responses of the ADC subtypes.

In the course of this study, 51 discrepancies were detected between the COH patient his-tology and follow-up pathology carried out under the auspices of CIRECA at U. Mass. Medi-cal School by a board-certified pathologist. The majority of these 51 discrepancies could be adjudicated by immunohistochemistry (IHC), using thyroid transcription factor 1 (TTF-1 for ADC) and p40 for SqCC. The SHP results were found to agree with IHC in most cases, indi-cating the sensitivity and value of SHP.

The results of this study demonstrate that SHP competes favorably with classical patholo-gy, augmented by IHC, in overall diagnostic accuracy.

References [1] A. Akalin, X. Mu, M. Kon, A. Ergin, S. Remiszewski, C. Thompson, D. Raz, B. Bird, M. Miljković,

M. Diem, "Classification of Malignant and Benign Tumors of the Lung by Infrared Spectral Histopathology (SHP)", Laboratory Investigation 95, 406-421 (2015)

Poster Session

Poster Session

Qualitative Analysis of Human Sebum in Sebaceous Glands in-situ Using FTIR Microspectroscopy

Agnieszka Banas1, Krzysztof Banas1, Bhimsen Rout2, Mei Bigliardi-Qi2, Paul L. Bigliardi2, Bryan Ho Siu-Yin2 and Mark B.H. Breese1

1Singapore Synchrotron Light Source, NUS; 5 Research Link, 117603 Singapore 2Institute of Medical Biology, A*Star; 8A Biomedical Grove, #06-06 Immunos,

138648 Singapore

Common skin disorders such as acne, folliculitis, hair loss, seborrhoic dermatitis, sebaceous gland hyperplasia or adenomas1 as well as very common dry, itchy skin in elderly are linked to changes in quantity and quality of sebum production and alteration in sebaceous gland activity. Sebaceous glands are connected to the hair follicle and therefore their pathophysiology is closely related to the pilosebaceous unit. A few recent papers have discussed the characterization of different parts of hair cuticle, medulla and cortex by means of FTIR microspectroscopy2. However, evaluation of quality of sebaceous glands and sebum composition on different areas (body, scalp, vertex, occipital) and various hair types (villus, scalp hair and miniaturized hair) in healthy and patients is not currently very well understood. A high photon flux and brilliance of synchrotron radiation enable FTIR experiments with high spatial resolution giving more insights into the composition of sebum in-situ in sebaceous glands without contamination with surface lipids and comparing this composition in various skin disorders and body sites. In the poster we will present some preliminary results obtained by a close collaborative effort and how this might affect the knowledge of sebum composition in normal sebaceous glands and compare in to sebum composition in hair loss and other hair disorders. These studies could also help to develop artificial sebum that is more natural and closer to human sebum and that can be used for disorders with too little sebum production, such as dry skin.

References [1] a) S. W. Youn, Clinics in Dermatol. 28, 8-11 (2010)

b) C. C. Zouboulis, Clinics in Dermatol. 22, 360-366 (2004).[2] J.-L.Bantignies, J. Cosmet. Sci. 51, 73-90 (2000).

Poster Session

Comparison of Work-flows for Spectral Data Pre-processing and Multivariate Statistical Analysis by Means of Open Source

Solutions: Orange (Python) and R Studio (R language)

Krzysztof Banas1, Agnieszka Banas1 , and Mark B. H. Breese1,2

1Singapore Synchrotron Light Source, National University of Singapore, 5 Research Link, Singapore 117603

2Physics Department, National University of Singapore, 2 Science Drive 3, Singapore 117542

Proper sample preparation, selection of correct sampling technique, tuning the parameters of the experiment and collecting the spectral data is very important phase in the analysis of biological samples. But it’s only the first step. Next, and crucial for achieving the goal of any project, is spectral data evaluation stage. Comparison of the work-flows for pre-processing, univariate and multivariate statistical analysis of the spectral data based on open source solutions: R Studio [1] (using R language) and Orange platform [2] (using Python language) is presented in this contribution. There are certain advantages of each work-flow discussed here in terms of user-friendliness, speed, scalability and flexibility as well as the option to implement machine learning and BigData algorithms.

References [1] R: A Language and Environment for Statistical Computing, R DevelopmentCore Team, Vienna, Austria

(2017) http://www.R-project.org [2] J. Demsar, T. Curk, A. Erjavec, C. Gorup, T. Hocevar, M. Milutinovic, M. Mozina, M. Polajnar, M. Toplak,

A. Staric, M. Stajdohar, L. Umek, L. Zagar, J. Zbontar, M. Zitnik, B. Zupan, “Orange: Data Mining Toolbox in Python”, Journal of Machine Learning Research 14(Aug), 2349-2353 (2013).

Poster Session

Infrared Analysis of Cystic Fibrosis (CF) Cell Models

Giuseppe Bellisola1, Sara Caldrer2, Mariangela Cestelli Guidi1, Gianfelice Cinque3, Silvia Vercellone2

1 INFN-LNF, via E. Fermi 40, 00044 Frascati, Italy

2 Department of Medicine, Cystic Fibrosis Translational Research Laboratory "D. Lissandrini", University of Verona, Italy

3 Diamond Light Source, MIRIAM beamline B22, Chilton-Didcot, Oxon OX11 0DE, UK

Background and aims. More than 2,000 different mutations have been identified in the Cystic Fibrosis Transmembrane conductance Regulator (Cftr) monogene [1]. Most of them are responsible of the autosomal recessive multi-organ disease Cystic Fibrosis (CF). This is directly and/or indirectly dependent on the impaired chloride anions and bicarbonate transport across cell membrane of secreting epithelia which is mediated by a 1480 amino acids ubiquitous transmembrane protein channel called CFTR. Cftr defects have been grouped into six classes [2] and the most frequently observed in CF patients are those impairing the production of full-length CFTR protein (class I) or affecting protein maturation and processing (class II). CFTR-modulating drugs developed to rescue the expression/functioning of CFTR are under evaluation in clinical trials with CF patients. A few drug have recently obtained the approval for the treatment of patients with particular Cftr genotypes [3]. Aimed at developing biomedical applications of Fourier transform (FT) infrared (IR) spectroscopy and microscopy (microFTIR) we performed IR analysis in CF and non-CF cell models in order to find out spectral markers useful for clinical diagnosis, ex vivo drug evaluation, and therapy follow up. Materials and methods. Model epithelial cells bearing Cftr-mutated and Cftr-corrected gene and peripheral blood lymphocytes from patients with different CF genotypes were analyzed in samples subjected to different experimental conditions. MicroFTIR was applied to dried cells deposited in a close monolayer on ZnSe window using synchrotron radiation (SR) or globar as IR sources to obtain FTIR absorbance spectra of single cells or groups of cells, respectively. Unsupervised multivariate PCA and classical supervised functional group analysis methods were applied to identify spectral patterns and IR markers. Results and conclusions. Consistent results were obtained in comparable samples probed by microFTIR performed with SR and globar as IR sources. PCA model identified combinations of variables within the amides region (1700-1500 cm-1) able to separate CFTR-defective from CFTR-corrected epithelial cells. Most significant variations involved the vibrations of α-helical and anti-parallel and aggregated β-sheets in proteins allowing to suggest that an excess of aggregated beta-sheets characterized CFTR-defective epithelial cells. The model reflected also spectral variations induced by CFTR-modulating drugs in cells. The same model was able to classify separately also CF lymphocytes according to the basic Cftr defects in patients.

Acknowledgement: The proposals SM9056 and SM8474 approved by Diamond Light Source Ltd received funding from the European Community's 7th Framework Programme (FP7/2007-2013) under grant agreement no.226716 References [1] Cystic Fibrosis Mutation Database available at www.genet.sickkids.on.ca/cftr [2] G. Veit, R.G. Avramescu, A.N. Chiang et al., Mol Biol Cell. 27, 424-33 (2016). [3] S.M. Rowe, A.S. Verkman, Cold Spring Harb Perspect Med. 3, a009761 (2013).

Experimental Designs for comparing Variance Contributions in Nested Data

Claudia Beleites1,2, Fabian Tietz3, Sascha Rohn3, Andrea Krähmer1

1 Julius-Kühn-Institut, Königin-Luise-Str. 19, Berlin, Germany,2 Chemometric Consulting and Chemometrix GmbH, Södeler Weg 19, Wölfersheim, Germany,

3 Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Hamburg, Germany

The CocoaChain project aims at establishing high quality along the processing chain of cocoa, andvibrational spectroscopy (mid-IR and NIR) are used to identify yeast strains and for quality control.GC-MS and HPLC-MS analyses are used as reference for spectroscopic calibration of variousconstituents of cocoa. In this poster, we compare two different sampling strategies (experimentaldesigns) for analyzing the importance of different sources of variance. Biospectroscopic data is typically subject to a variety of sources of variation ranging from purelybiological variance (strain-to-strain, patient-to-patient, variance between biological replicates, etc.)to sources of analytical error (sampling error, variance between technical replicates, measurementnoise, ...). Here, we focus on random variation (variance) rather than systematic influence (bias).Often, these sources of variance form a naturally nested structure following the experimentalprocess (fig. 1), e.g., a cocoa fermentation is done using a particular yeast strain, of eachfermentation a number of primary samples may be taken in the field*, which are in turn divided intoaliquots for analysis in the lab. The spectroscopic configuration may not measure the wholespecimen, leading to measurement spots (locations) being nested in the aliquots, and finally,repeated measurements reveal measurement noise. In general, experiments are efficient if the effort is concentrated on large contributors of variance.Measuring the different variance contributions is therefore highly important. The more so, as itallows to drop unimportant variance contributors during subsequent stages of the experiment. Thisin turn allows to drastically reduce the number of measurements without compromising the qualityof the study. While classical fully nested measurements are feasible for spectroscopy (with anautosampler, fig. 2), reference analyses are more costly so we employ a staggered nested design(fig. 3). The staggered design offers a more uniform distribution of degrees of freedom over theexperimental levels with much fewer measurements but requires more complex statistical analysis.

*If these primary samples are taken e.g. at different times of the fermentation, time and fermentation would be crossed,not nested. However, here we consider primary samples taken at the same time in order to measure sampling error.

AcknowledgementsFinancial support of the project “CocoaChain” (IGF 169 EN/3) by the AIF (Arbeitskreis industirelle Forschung) andFEI (Forschungskreis der Ernährungsindustrie) is highly acknowledged.

primary sample

aliquot

fill

NIRNIR NIRNIRNIRNIRNIRNIR

NIRNIRNIRNIR

stir stirfill stirfill

aliquotaliquot

primary sample

aliquot aliquotaliquot

sub- sample

sub- sample

sub- sample

sub- sample

GC-MS GC-MS GC-MS GC-MSGC-MSmeasurement noise

measurement volume/location

subsample/aliquot

primary sample

fermentation/biol. replicate

strain

Figure 2: Fully nested DoE leads to many measurements at the lower levels. This is feasible for spectros- copy (with autosampler) .

Figure 1: CocoaChain nested data. The first two levels (grey) cannot be characterized yet: at the moment, only one fermen- tation is available.

Figure 3: Staggered nested DoE has only one duplicate at each level, leading to much fewer measurements on the lowest level and an even distribution of degrees of freedom over the levels.

AnDa

3C

7Te

The higrecognitirespectivbenefits destructicontrast-potentialvivo. Ousystem efour keabsorbanbest mettissue asresected tumor dethe measDetailedmultivarspectral bioliquidprovidedthe colon

In additi

sensitiviprobe wand mon

Table 1 Co

Method 

MIR 

Fluo 

MIR|Fluo

In Com

Olganastasya Manielyan,1O

2SCenter for R

chnical Uni

ghly selectivion of variove mixture c

with the iveness and n-enhancing al for variousur developmenables to teey spectrosnce, 6diffusethod (or theirs it´s neededabdominal t

etection withsurements on

d comparisonriate data anamethods hav

ds, such as bd the best indn.

ion, diagnos

ty, Sps is spas developed

nitoring of tu

omparison of spectr

Preprocessin

2D 

none 

o  SNV|2D+SN

MIR-fibmpetition w

a BibikovaMelentevaOlaf Mine

1araratov Nati

Radiology CU

4Samara iversity of B

8

ve FT-IR sus organic m

constituents. opportunitie

non-invasiveagents of tiss types of the

ment of unist various si

scopy methe NIR-reflectr best combid for cancertissues with ch other specn tissue sampn of accuracyalysis of specve been appl

blood and uridicate the mo

stic capabilit

pecificity. Bad as shown o

umor spread,

roscopic methods fo

ng  FP  FN 

5  6 

22  19 

V  1  1 

ber Spectrwith Ram

or in

a,1,2 Urszua,4 Iskandeet,3Andrey

rt photonicsional Resear6, Medical

UniversitätsmState Techn

Berlin, Insti8global mod

spectroscopymolecules anCombination

es of fiber eness as wellsue provides e human canique Multi-ingle and comhods: Ramation, and fluonation) for ar surgeries. Ocancers is prtroscopic meples in all my, sensitivityctroscopic dalied to analyine, of patienorphological

ties of fluore

ased on obtaion Figure 1.determine su

or kidney cancer dia

%Acc  %Sns 

88  88 

55  63 

99  98 

roscopy foman, NIR-r

Combina

ula J. Zabaer Usenovy Bogomo

s GmbH, Berch State UnPhysics and

medizin, Benical Univeritute for Optdelling, Aale

y is capablend quantifican of FTIR sp

optics, incl as absence a significan

ncerex-vivo aSpectral Fibmbined fiberan scatterinorescence – ta detection ofOur recent rresented to cethods. In or

methods werey and specifata to build pyse not only nts before an

differences

escence and

ined results, The probe auccessful dia

agnostics 

%Spc 

88 

46 

98 

r Tumor Sreflection,

ation?

arylo,3 Val,1,5 Tatianlov,4,6 Via

erlin, Germaniversity, Sd Optical Dierlin Germanrsity, Samartics & Atomen, German

e of exact ation of the pectroscopy cludingnon-of extrinsic

nt diagnostic and even in-ber (MSF-) r probes for ng, FTIR-to select the f malignant research of ompare the Frder to incre performed i

ficity for tissprediction mbiopsies of

nd after surgebetween the

d FTIR spectinvestigatedand malignpatients wsynergic gFTIR-ATRfor kidney Table 1, wfalse negacombined F

allows to coagnosis and r

Sensing: , Fluoresc

leria Belikna Sakharoacheslav A

any aratov, Rusiagnostics, ny ra, Russia

mic Physics ny

FTIR absorbease the datain the same psues determi

models for eachealth and m

ery. Exemplahealthy and

troscopy comd using matcnant biopsywith kidnegain of com

R and fluorecancer diag

where FP is ative, Acc iFTIR-ATR aontribute to threalistic canc

Figure 1.Pthe combinfluorescen

Poster S

cence –

kova,4 ova,1 GeorArtyushenk

ssia CBF, Chari

(IOAP), Be

bance opportua informationpreselected pination were ch method.Mmalignant tisary, FTIR-ab

d malignant r

mbination hching pairs o

y samples oey cancer.Ombining fib

escence specgnostics presfalse positivis accuracy,

and fluorescehe efficient

cer prognosis

Principal schemned FTIR-AT

nce fiber probe

Session

rgy ko1

ité-

erlin

unities of n content positions.

done by Moreover, ssues, but bsorbance egions of

ave been of normal of a few Observed ber-based ctroscopy sented in ve, FN is , Sns is

ence fiber detection s.

me of R and e

Poster Session

Microscopy-based Raman Spectroscopy of Fungal Melanins in a Genetically Amenable Ascomycete

Maria Dittrich1, Heinz Sturm2, Vladimir Zaitsev4, Carlos Paulo1, Romy

Breitenbach2,3, Jörg Toepel2, Nicole Knabe2, Anna A. Gorbushina 2,3

1 University of Toronto, 1265 Military Trail, Toronto ON, M1C 1A4, Canada 2 BAM, Unter den Eichen 87, 12205 Berlin, Germany

3 Freie Universität Berlin, Malteserstrasse 74-100, Berlin, Germany 4 Moscow State University, Leninskie Gory, 1-2, Moscow, Russia

Fungal melanins are distinctive markers of animal (including human) and plant pathogenic fungi as well as their environmental relatives. These complex polyphenols play important roles in pathogenicity and stress tolerance while being essential components of fungal cell walls and useful biomarkers. Accordingly, it is important to clarify melanin function in black yeasts, a group of clinical and environmental importance [1].

Here we report signatures of melanins and carotenoids in single and double knockout strains of the model environmental black yeast Knufia petricola A95 [2]. This genetically amenable strain is an ancestor of many plant and animal pathogenic fungi. Knock-out mutants of protective pigment genes KpPKS (polyketide synthase), KpSDH (scytalone dehydratase) and KpPDG (phytoene desaturase) were studied using Raman spectroscopy.

Mutants allow discrimination between the various pigments and elucidation of melanin structure. Hence interactions between natural fungal melanins (as well as other protective pigments) and complex environmental/material matrices can be characterised on a range of spatial and temporal scales. A library of Raman spectra of natural fungal melanins will be created to serve as an exploration tool to detect and study pathogenic and environmental fungi in clinical samples and on material surfaces, especially in extreme environments.

We used an NTEGRA Spectra system from NT-MDT (Zelenograd, Moscow 124460, Russia) that combines atomic force microscopy (AFM) and Raman microscopy. The system is equipped with an inverted optical microscope, an upright optical microscope and laser beams of 532, 633 and 785 nm wavelengths. Individual Raman spectra were obtained for reference substances, mutant strains and the wild-type strain. In addition, we applied tip- and surface- enhanced techniques [3] to collect Raman spectra. References [1] G. S. de Hoog, V. A. Vicente, A. A. Gorbushina, Mycopathologia 175, 365–368 (2013). [2] S. Noack-Schönmann, T. Bus, R. Banasiak, N. Knabe, W.J. Broughton, H. Den Dulk-Ras, P. J. J. Hooykaas,

A. A. Gorbushina, AMB Express 4, 80 (2014). [3] C. Paulo, M. Dittrich, Journal of Raman Spectroscopy 44, 1563-1569 (2013).

Figure 1. Raman spectra of synthetic melanin, wild type strain Knufia petricola A95 and its mutants

Poster Session

High-Contrast Brillouin and Raman Micro-spectroscopy for Simultaneous Mechanical and Chemical Investigation of Microbial Biofilms

Debora Casagrande Pierantoni1, Laura Corte1, Luca Roscini1, Sara Mattana3,

Daniele Fioretto2,3, Gianluigi Cardinali1,2

1Department of Pharmaceutical Sciences, University of Perugia – Italy,

2CEMIN Excellence Research Center – University of Perugia, 3Department of Physics and Geology- University of Perugia – Italy,

Biofilm forming microbial cells characterize for their ability to grow onto solid surfaces embedded in a polymer matrix of eso-polysaccharides (EPS) and their resistance to the actions of many chemical and physical agents. In particular, biofilm forming yeast species represent both a threat for human health and a problem in food industry. The importance of biofilms of fungal, bacterial and mixed origin depends on their increased resistance to antibiotics, anti-fungal drugs and extreme conditions. The mechanical characteristics of the biofilm, although are poorly studied, appear of primary interest to elucidate the mechanisms governing the stability and the dispersion of the cells involved in the biofilm. Hence the importance to study biofilms. Candida strains selected for their different ability to form biofilms were grown on top of aluminum foil and stainless steel washers. Brillouin-Raman micro-spectroscopy (BRMS) performed with a new tandem Fabry-Perot interferometer was applied to map samples of Candida biofilms with the aim of obtaining proof of principle on the possibility to characterize mechanical (viscoelastic) and chemical properties of biofilms at high resolution [1, 2]. Chemo-mechanical maps of Candida biofilms were obtained without the need of staining or touching the sample. Correlation studies between Raman and Brillouin data suggested the role of both extracellular polymeric substances and of hydration water in inducing a marked local softening of the biofilm. In particular, BRMS allowed establishing the different water content in C. albicans biofilm as compared to C. parapsilosis and C tropicalis biofilms. The sensitivity of the method and the possibility to perform correlation studies with minimal analytical variability in the sample open the way to compare the ability of different microorganisms to survive in inhospitable environments and develop resistance to antibiotics. Overall, we obtained proof-of-concept that joined micro-Raman and micro-Brillouin techniques allow exploring the physical and chemical properties of biofilms at high resolution in the same sample area, concurrently. This contactless detection method to characterize mechanical (viscoelastic) and chemical properties of biofilms enables the comparison of microbial biofilms. References [1] F. Palombo, M. Madami, N. Stone and D. Fioretto, Analyst 139, 729-33 (2014). [2] S. Mattana, M. Alunni Cardinali, S. Caponi, D. Casagrande Pierantoni, L. Corte, L. Roscini, G. Cardinali and

D. Fioretto, Biophys Chem. S0301-4622, 30175-8 (2017).

Poster Session

Evaluation of T-Cell Activation with Raman Microspectroscopy

Neha Chaudhary1,2*, Aidan D. Meade1,2

1School of Physics, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

2 Radiation and Environmental Science Centre, Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

Inflammation is a characteristic of various degenerative diseases, including cancer, and involves activation of T-cells [1]. While many clinical methods to diagnose inflammation exists but still no single, specific and label free methods is available. Developing a rapid, non-invasive and label free methodology for detecting the activation of T-cells will radically improve both the diagnosis and the prognosis in patients with the condition [2]. Raman spectroscopy has proven to be a reliable diagnostic tool for detecting cellular physiology. The unique biochemical fingerprint of cells can provide quantitative and qualitative understanding about the changes in the biochemical components of the sample under investigation. This in-vitro study was conducted to investigate the effect of inflammatory activation on a T-cell line. Jurkat cells were treated with phytohemagglutinin (PHA) at numerous concentrations to induce inflammation.The viability of cells was then evaluated using 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT). Parallel reference measurements of activation were performed on cells fluorescently labeled with anti-CD69 using a plate reader. Jurkat cells treated with the optimized PHA concentration and untreated were then fixed on calcium fluoride slides for spectroscopic measurements. The effect on CD69 expression of activated cells was also measured using flow cytometry in parallel to Raman spectroscopy. This study is intended to present a unique Raman signature for T-cell activation and to demonstrate the potential of vibrational spectroscopy as a specific and label-free technique for detection of activated T-cells. References [1] A. Ramoji et al., “Toward a Spectroscopic Hemogram: Raman Spectroscopic Differentiation of the two Most

Abundant Leukocytes from Peripheral Blood”, Anal. Chem. 84(12), 5335-5342 (2012). [2] A. Weselucha-Birczynska, M. Kozicki, J. Czepiel, M. Birczynska,”Raman Micro-spectroscopy Tracing

Human Lymphocyte Activation”, Analyst 138, 7157-7163 (2013).

    Poster Session

A Compact Raman Imaging System for Bladder Tissue Analysis

Eliana Cordero*1

, Iwan W. Schie1, and Jürgen Popp

1,2

1Leibniz Institute of Photonic Technology (IPHT), Albert-Einstein-Straße 9,

Jena, Germany 2Institute of Physical Chemistry, Friedrich Schiller University Jena,

Helmholtzweg 4, 07743 Jena, Germany

New modalities in detection and management of bladder tumours for early bladder cancer diagnosis still depend of invasive and time consuming procedures, such as the extraction of biopsies, following a pathological analysis of the samples [1, 2]. There is a significant need for non-invasive and rapid methods for in vivo bladder cancer diagnostic. A large number of in vivo and in vitro experiments have demonstrated the ability of Raman spectroscopy to diagnose oncological diseases by providing label-free biochemical information from tissue samples [3-5]. Recent in vivo investigations have shown promising results, but were limited by long acquisition times and high auto-fluorescence backgrounds, due to the nature of the samples [6, 7]. A more in-depth characterization of bladder biopsies is needed and new computational methods to preprocess and analyze the measured Raman signals are required [8]. Therefore, this work is aimed to develop a small and compactRaman fiber probe based imaging system to characterize large fresh bladder tissue biopsies. Moreover, we present a comprehensive study on the evaluation of background correction strategies for Raman spectra of highly fluorescent bladder tissues. We compare theoretically and experimentally an instrumental approach, i.e. shifted Excitation difference Raman spectroscopy (SERDS), with a computational approach, i.e. extended multiplicative scattering correction (EMSC), for background correction. Acknowledgment: This work is supported by the EU-funded project MIB (No 667933)

References [1] O. W. Darren, B. K. Beharry, D. Wetherell. N. Papa, M. Weerakoon, A. Sliwinski, M. Brausi, D. Bolton, N.

Lawrentschuk, “New Technologies for Diagnosis of Non-Muscle-Invasive Bladder Cancer (NMIBC) and its Management”, J IntegrOncol 3 (2014).

[2] R. Colombo, R Naspro, P. Bellinzoni, F. Fabbri, G. Guazzoni, V. Scattoni, A. Losa, P. Rigatti , “Photodynamic diagnosis for follow-up of carcinoma in situ of the bladder”, TherClin Risk Manag 3, 1003-1007 (2007).

[3] J. T. Motz, M. Hunter, L. H. Galindo, J. A. Gardecki, J. R. Kramer, R. R. Dasari, M. S. Feld, “Optical fiber probe for biomedical Raman spectroscopy“, Appl. Opt. 43, 543-553 (2004).

[4] W. Wang, J. Zhao, M. Short, H. Zeng, “Real-time in vivo cancer diagnosis using Raman spectroscopy”, J Biophotonics 8, 527-545 (2015).

[5] K. Kong, C. Kendall, N. Stone, I. Notingher, “Raman spectroscopy for medical diagnostics- From in-vitro biofluid assays to in-vivo cancer detection”, Adv, Drug Deliv. Rev. 89, 121-134 (2015).

[6] E. Cordero, F. Korinth, C. Stiebing, C. Krafft, I. Schie, J. Popp, “Evaluation of Shifted Excitation Raman Difference Spectroscopy and Comparison to Computational Background Correction Methods Applied to Biochemical Raman Spectra“, Sensors 17 (2017).

[7] R. Gautam, S. Vanga, F. Ariese, S. Umapathy, “Review of multidimensional data processing approaches for Raman and infrared spectroscopy”, EPJ Tech. Instrum. 2 (2015).

[8] P. J. Cadush, M. M. Hlaing, S. A. Wade, S. L. Mxarthur, P. R. Stoddart, “Improved methods for fluorescence background subtraction from Raman spectra”, Mater. Sci. 44, 1587-1595 (2013).

 

Poster Session

Merging FT-IR and NGS for Simultaneous Phenotypic and Genotypic Identification of Pathogenic Candida Species

Laura Corte 1, Claudia Colabella 1, Luca Roscini 1, Volha Shapaval2, Achim

Kohler 2, Valeria Tafintseva 2, Carlo Tascini 3 and Gianluigi Cardinali 1,4

1 Department of Pharmaceutical Sciences - Microbiology, University of Perugia, Perugia (Italy)

2 Department of Mathematical Sciences and Technology, Norwegian University of Life Sciences, 1432 Ås, Norway

3 Azienda Ospedaliera dei Colli - Ospedale Cotugno, Napoli, Italy 4 CEMIN, Centre of Excellence on Nanostructured Innovative Materials - Department of

Chemistry, Biology and Biotechnology - University of Perugia, Perugia, Italy The rapid and accurate identification of pathogen yeast species is crucial for clinical diagnosis due to the high level of mortality and morbidity induced, even after antifungal therapy. For this purpose, new rapid, high-throughput and reliable identification methods are required. We described a combined approach based on two high-throughput techniques in order to improve the identification of pathogenic yeast strains. Next Generation Sequencing (NGS) of ITS and D1/D2 LSU marker regions together with Fourier Transform Infrared Spectroscopy (FTIR) were applied to identify 256 strains belonging to Candida genus isolated in nosocomial environments. Multivariate data analysis (MVA) was carried out on NGS and FT-IR data-sets, separately. Strains of Candida albicans, C. parapsilosis, C. glabrata and C. tropicalis, were identified with high-throughput NGS sequencing of ITS and LSU markers and then with FTIR. Inter- and intra-species variability was investigated by Consensus Principal Component Analysis (CPCA) which combines high-dimensional data of the two complementary analytical approaches in concatenated PCA blocks normalized to the same weight. The total percentage of correct identification reached around 97.4% for C. albicans and 74% for C. parapsilosis while the other two species showed lower identification rates. Results suggested that the identification success increases with the increasing number of strains actually used in the PLS analysis. The absence of reliable FT-IR libraries in the current scenario is the major limitation in FTIR-based identification of strains, although this metabolomics fingerprint represents a valid and affordable aid to rapid and high-throughput to clinical diagnosis. According to our data, FTIR libraries should include some tens of certified strains per species, possibly over 50, deriving from diverse sources and collected over an extensive time period. This implies a multidisciplinary effort of specialists working in strain isolation and maintenance, molecular taxonomy, FTIR technique and chemo-metrics, data management and data basing.

Poster Session

Raman Micro-spectroscopic Identification of Streptococcus pneumoniae Differentiated from other Streptococcus species

Marcel Dahmsa,b,c, Simone Eiserlohb,ShuxiaGuoc,d, Thomas Bocklitzc,d,

Jürgen Rödele, Jürgen Poppa,b,c,d,, Ute Neugebauera,b,c,d

aInfectoGnosticsResearch Campus Jena, Center for Applied Research, Jena, Germany b Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany

c Leibniz Institute of Photonic Technology, Jena, Germany d Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University

Jena, Jena, Germany eInstitute of Medical Microbiology, Jena University Hospital, Jena, Germany

Streptococcus pneumoniae is the most occurring pathogen in community-acquired pneumonia (CAP), the most death-causing infectious disease worldwide [1]. Since an early start of adequate therapy is highly important for patient’s outcome, a fast and reliable identification of the infection causing pathogen is necessary. Currently, the standard procedure includes cultivation and additional chemical testing, e.g., reaction against optochin or bile and takes between 24 and 72 hours [2]. Other Streptococcus species occur as tracheal flora. They are normally not the cause of a pneumonia infection and therefore should be excluded while identification. Here, we report the application of Raman spectroscopy for principally differentiation of S. pneumoniae from other Streptococcus species as first step to a fast pneumonia diagnosis.

For a reliable detection of S. pneumoniae Raman spectra (see Fig. 1) were taken from 5 Streptococcus species (eight S. pneumoniae strains, 4 other Streptococcus species) after cultivation on blood agar and preparation on CaF2 slides. Data analysis was done with the free statistical software R using support vector machines (SVM) for classification.

The chemometric model performs with high sensitivity and specificity for the differentiation of S. pneumoniae and other Streptococcus species. This culture-based experiment indicates the general differentiation potential and paves the way for investigations on pathogens isolated directly from lung body fluids, e.g., sputum or bronchoalveolar lavage (BAL), which would save valuable time currently needed for cultivation.

Acknowledgements: The financial support by the Federal Ministry of Education and Research (BMBF), Germany via the project “InfectoGnostics” (13GW0096F) and the Integrated Research and Treatment Center “Center for Sepsis Control and Care” (CSCC, FKZ 01EO5002) is highly acknowledged. References [1] M. W. Pletz et al., F1000Research 2016, 5(F1000 Faculty Rev):300 [2] B. Spellerberg and C. Brandt, in: J. Versalovic, K. Carroll, G. Funke, J. Jorgensen, M. Landry, D. Warnock

(eds.): “Manual of Clinical Microbiology”, 10th Edition, Streptococcus, p. 331-349, ASM Press, Washington, DC (2011).

Figure 1.Raman spectra of different Streptococcus species

Poster Session

Synchrotron Infrared Microspectroscopy as a Tool for Probing Drug-cell Interactions in Living Biological Cells

Joanna Denbigh3, JamesDoherty2,3, Zhe Zhang2, Katia Wehbe2,

Gianfelice Cinque2 and Peter Gardner3

1 School of Environment and Life Sciences, University of Salford, Cockroft Building, Salford, M5 4WT

2Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE

3Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN

Synchrotron Fourier transform infrared microspectroscopy is a widely used tool for differentiating between cellular responses to different drugs. [1-5] Analysis of fixed, dried renal cell carcinoma (RCC) cells at the Diamond Light Source synchrotron has demonstrated an ability to separate the

response of drug-resistant cells to different doses of the chemotherapeutic drug paclitaxel (see Fig.1), as well as distinguish between drug-resistant and wildtype cells prior to treatment. The information available from fixed, dried cells is, however, ultimately limited by spectral effects of fixation, which vary depending on the fixation method used. [6] It is therefore desirable to analyse cells in their natural, hydrated state. This also has the advantage of reducing the resonant Mie scattering effects that can hamper dried cell spectra.[7]The study of living cells in an aqueous environment introduces strong water absorbance into spectra. We have developed and tested a water correction algorithm which allows for a significant increase in spacer thickness from 6 to 12 micron, thus reducing shear stresses on cells during analysis. Having tested this methodology on fixed cells in aqueous suspension, we also demonstrate its use in the study of live K562 acute myeloid leukaemia (AML) cells, treated with

two novel chemotherapy agents. This method retains sufficient spectral detail to differentiate between samples based on the different modes of action of the two novel agents. References [1] A. B. de Carvalho et al., "Chemotherapeutic response to cisplatin-like drugs in human breast cancer cells

probed by vibrational microspectroscopy", Faraday discussions (2016). [2] J. L. Denbigh et al., "Probing the action of a novel anti-leukaemic drug therapy at the single cell level using

modern vibrational spectroscopy techniques", Scientific Reports 7 (2017). [3] J. Doherty, G. Cinque, and P. Gardner, "Single-cell analysis using Fourier transform infrared

microspectroscopy", Applied Spectroscopy Reviews 52(6), 560-587 (2017). [4] K. R. Flower et al., "Synchrotron FTIR analysis of drug treated ovarian A2780 cells: an ability to

differentiate cell response to different drugs?" Analyst 136(3), 498-507 (2011). [[5] C. Hughes et al., "Investigating cellular responses to novel chemotherapeutics in renal cell carcinoma using

SR-FTIR spectroscopy", Analyst 137(20), 4720-4726 (2012). [6] E. Gazi et al., "Fixation protocols for subcellular imaging by synchrotron‐based Fourier transform infrared

microspectroscopy", Biopolymers 77(1), 18-30 (2005). [7] P. Bassan et al., "FTIR microscopy of biological cells and tissue: data analysis using resonant Mie scattering

(RMieS) EMSC algorithm", Analyst 137(6), 1370-1377 (2012).

Fig. 1. Canonical variate analysis showing separation between resistant control cells (black) and cells treated with low and high doses of paclitaxel (green and blue respectively).

Poster session

Hierarchical classification of variations in grass pollen quality using MALDI-TOF MS

Sabrina Diehn1, Boris Zimmermann2, Murat Bağcioğlu2 , Stephan Seifert1,3, Siri

Fjellheim2, Mikael Ohlson2, Achim Kohler2, Steffen Weidner3, Janina Kneipp1,3

1Humboldt-Universität zu Berlin, Institut für Chemie, Brook-Taylor-Straße 2, 12489 Berlin, Germany

2Norwegian University of Life Sciences, Drøbakveien 31, 1432 Ås, Norway 3Bundesanstalt für Materialforschung und –prüfung, Richard-Willstätter-Straße 11, 12489

Berlin, Germany

Pollen grains are the carriers of male genetic material and thus should be as robust as possible with regard to environmental stress. Environmental factors, such as temperature and precipitations, may influence the chemical composition of pollen as well as its germination and fertilization rates. Variances in the chemistry of complex biological systems such as pollen grains can be characterized using spectroscopy or spectrometry and chemometric methods. We and others have shown that pollen grains have a species-specific and environment-specific chemical composition which can be identified based on their vibrational and mass spectra1, 2. In addition, physiological influences, e.g., in germinating pollen grains can be detect and characterized with chemometric tools. These examples demonstrate that biological data sets are often organized in a hierarchical structure following, for example phylogenetic relationships, individual differences or histological substructures in the pollen tissue3, 4. Using Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), species-specific peak patterns can be obtained from pollen grains1. Assuming that spectral similarity follows a hierarchy, high sensitivity and specificity of the analytical and chemometric methods is required. In our study, MALDI-TOF MS in combination with partial least squares regression (PLS-R) was applied in order to identify variances in species, population and specific growth conditions of plants simultaneously. The role of environmental changes on pollen quality of three different grass pollen species will be discussed. The combined information provides insight into the evaluation of pollen quality based on the chemical fingerprint represented in the MALDI spectra. The results may have impact in the broader field of environmental science including plant biology and agriculture research.

[1.] S. Seifert, S. M. Weidner, U. Panne and J. Kneipp, Rapid Commun. Mass Spectrom., 2015, 29, 1145-

1154. [2.] M. Bagcioglu, A. Kohler, S. Seifert, J. Kneipp and B. Zimmermann, Methods Ecol. Evol., 2017, 8, 870-

880. [3.] F. Schulte, J. Lingott, U. Panne and J. Kneipp, Anal. Chem., 2008, 80, 9551-9556. [4.] M. Joester, S. Seifert, F. Emmerling and J. Kneipp, Journal of Biophotonics, 2016, DOI:

10.1002/jbio.201600011

Poster Session

Microbial Single Cell Detection with Raman Spectroscopy: Taxonomic Resolution and Data Accuracy

Martina Alunni Cardinali1, Debora Casagrande Pierantoni2, Silvia Caponi3,

Gianluigi Cardinali2,4 and Daniele Fioretto1,4

1 Department of Physics and Geology- University of Perugia – I 2 Department of Pharmaceutical Sciences – University of Perugia – I

3.Istituto Officina dei Materiali del CNR(CNR-IOM) – Unit of Perugia -I 4CEMIN Excellence Research Center University of Perugia – I

Micro-spectroscopy by means of FTIR or Raman gives very promising perspectives in microbiology among which the possibility to analyze a sample without prior growth [1-3]. This condition offers the advantages of: (i) shorter analytical times, due to the lack of the 24-48 h necessary for growth. This

aspect can be crucial when the species identification is part of diagnostic process, normally requiring short times.

(ii) possibility of detecting VNC (viable non culturable) microorganisms that grow in the natural condition, but not in laboratory media.

(iii) unbiased estimation of the alpha-diversity, which can be significantly altered during the growth due to the different growing rates of the various strains and species.

However, this technique is challenged by the difficulty of obtaining reliable spectra from single cells, due to their inherent variability. Moreover, eukaryotic cells, as fungi, algae and protozoa have the additional inconvenience of undergoing a four phase life cycle, meaning that the metabolome of the same cell in each phase can be significantly different. In order to define the level of variability among different measures of the same cells, with a Raman microspectroscopy apparatus, we developed a simple taxonomic model consisting of four certified strains belonging to four pathogenic speciesCandida albicans, C. tropicalis, C. parapsilosis and C. glabrata. Cells where grown with and without shaking and spectrawere recorded in both polarized and non-polarized configuration. Twelve independent readings for each combination of strain and experimental condition were carried out. Two different analytical approaches were employed, one considering the distance of the spectra from the most representative (central) spectrum of the set and one calculating the distance of the spectra from the average spectrum. All analyses were performed for the whole spectrum and for the different spectral regions separately. Results showed that indeed a great level of variability can be detected among the spectra of the same strain and that sometimes this variability is comparable with the distance between cells of different species. This condition reduces the taxonomic resolution (i.e. the possibility to discriminate between species) and requires a number of countermeasures that will be presented and discussed.

References [1] F. Palombo, M. Madami, N. Stone and D. Fioretto, Analyst139, 729-33 (2014). [2] S. Mattana, M. Alunni Cardinali, S. Caponi, D. Casagrande Pierantoni, L. Corte, L. Roscini, G. Cardinali and

D. Fioretto, BiophysChem. S0301-4622, 30175-8 (2017). [3] F. Scarponi, S. Mattana, S. Corezzi, S. Caponi, L. Comez, P. Sassi, A. Morresi, M. Paolantoni, L. Urbanelli,

C. Emiliani, L. Roscini, L. Corte, G. Cardinali, F. Palombo, J. R. Sandercock, and D. Fioretto, Phys. Rev. X 7, 031015 (2017).

    Poster Session  

Vibrational Spectroscopy as a High Throughput Technique for Bacteria Identification

Angela Flacka,b*, Ganesh D. Sockalinguma**, Matthew J. Bakerb***

aUniversité de Reims Champagne-Ardenne, MéDIAN-Biophotonique et Technologies pour la

Santé, UFR de Pharmacie, Reims, France b West CHEM, Department of Pure and Applied Chemistry, Technology and Innovation

Centre, 99 George St, University of Strathclyde, Glasgow, G1 1RD, UK *Email: angela.m.flack@strath.ac.uk

**Email: ganesh.sockalingum@univ-reims.fr ***Email: matthew.baker@strath.ac.uk

Bacteria can threaten mankind in several different ways, notably in terms of infection and from use as a biological warfare agent.

Biology has been manipulated to be used as a weapon throughout history, recently, the 2001 Anthrax Letters case highlighted the ever evolving threat of biological warfare agents, they pose such a threat due their ease of manufacture, small doses required for maximum damage, and difficulty to track(1).

Hospital acquired infections are an increasing issue for healthcare services, time taken to culture and to identify the specific bacterial infection can risk incorrect antibiotic usage, adding to the global issue of antimicrobial resistance (AMR)(2).

Both of these threats from bacteria share the same issue when it comes to identification. During an attack being able to quickly identify which bacterium was used would lead to faster/specific support to victims, as well as providing rapid information to aid front line personnel. Within a hospital environment being able to identify the bacterium rapidly allows specific antibiotics to be prescribed rather than broad scale antibiotics.

This paper reports the use of a novel method of high throughput vibrational spectroscopy to demonstrate the use of ATR-FTIR for analysis of bacteria. This work will present a bacteria study involving more than 80 samples with the aim of advancing ATR-FTIR into a more high throughput, rapid approach to bacterial identification. References [1] V. Barras, G. Greub, “History of biological warfare and bioterrorism”, Clin Microbiol Infect 20, 497-502

(2014). [2] Raza, Sobia, “Healthcare Associated Infections”, SPICe Briefing (2011).  

Poster Session

Surface-enhanced Raman Spectroscopy for Therapeutic Drug Monitoring in Oncology: A Study on Sample Preparation

Stefano Fornasaro, Alois Bonifacio and Valter Sergo

Department of Engineering and Architecture, University of Trieste, Trieste; Italy

Reproducible quantification of drugs using surface-enhanced Raman spectroscopy (SERS) is currently drawing considerable attention in the research field of therapeutic drug monitoring (TDM) for cancer therapy, due to the possibility to provide analyses of drugs in body fluids within few minutes, with comparable or smaller errors with respect to routine TDM methods [1].

However, clinical samples are complex mixtures containing lipids and proteins that can bind the drug of interest; thus only a fraction of the drug molecules will be free to interact to the metal surface, and, hence, to benefit from the signal enhancement. Sample preparation methods andexperimental conditions must be carefully evaluated to obtain interpretable and comparable results.

Here we report the development of a standardized preanalytical method for two chemotherapeutic drugs, imatinib and irinotecan, both exhibiting strong affinity to plasma proteins.

To find the most efficient method for sample preparation, we analyzed different protein precipitation methods as well as ultrafiltration, for both reproducibility and reduction of matrix effect. References [1] A. Jaworska, S. Fornasaro, V. Sergo, A. Bonifacio, Biosensors 6(3), 47 (2016).

Poster Session

Sample Preparation for Bacteria Identification with Raman Spectroscopy

Cristina García-Timermans1*, Dmitry Khalenkow2, Benjamin Buysschaert1, Frederiek-Maarten Kerckhof1, Peter Rubbens3, Andrea Skirtach2, Nico Boon

1Center for Microbial Technology and Ecology (CMET), Ghent University,

Coupure Links 653, B-9000 Ghent, Belgium 2KERMIT, Department of Mathematical Modelling, Statistics and Bioinformatics,

Ghent University, Coupure Links 653, B-9000 Ghent, Belgium 3Laboratory of Nano and Biophotonics, Ghent University, Coupure Links 653,

B-9000 Ghent, Belgium

Gathering information about cellular communities and their environment can help for a deeper understanding and management of ecosystems. Rapid identification of cells is possible thanks to multiparametric tools such as Raman spectroscopy. This technique makes a unique fingerprint for each bacterial cell based on their molecular expression profile. Thus, we can cluster cells and simultaneously pinpoint molecular traits related to biological activity, which are explanatory for the clustering result. The sensitivity of Raman spectroscopy calls for a standardized sample preparation. Otherwise, results could lead to an incorrect classification. This work focuses on the development of a protocol for identifying label-free bacteria using Raman spectroscopy. Our findings demonstrate how results can be affected by such procedures as the storage of a sample, the drying time on the slide or extra steps of resuspension and centrifugation. In this work we propose a workflow to assure data reproducibility as sample preparation created artificial (technical) subpopulations. Users should be aware of the need for standardized sample preparation and recording of metadata, especially when looking into small spectral variations.

Poster Session

FT-IR Microspectroscopy of Cancer Cells and Extracellular Vesicles

M. Grube1, K. Shvirksts1, S. Kokorevicha2, E. Zandberga3, A. Abols3, A. Line3

1Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia 2State Forensic Science Bureau, Ministry of Justice of the Republic of Latvia, Riga, Latvia

3 Latvian Biomedical Research and Study centre, University of Latvia, Riga, Latvia

Nevertheless, that cancer cells abnormally secrete large quantities of extracellular vesicles (EV), it is a time-consuming and relatively expensive process to collect samples with high enough concentration to record qualitative FT-IR spectra. Sample preparation for IR-spectroscopy is another principally important step and option. In our previous studies, EV of cancer-derived cells were analyzed using FT-IR spectra recorded using HTS-XT. It was shown that spectrum of HEPES buffer overlaps with that of cells or EV, and thus proteins of cells or EV cannot be identified properly. Therefore, cells or EV were suspended in PBS buffer but 0,9% NaCl also can be used. The aim of this study was to find a method for FT-IR spectroscopy analysis of significantly lower counts of cells or EV. Under study were colorectal cancer cell lines derived from a primary - SW480, and metastatic - SW620, tumour cultured under hypoxic or normoxic conditions and their derived EV. Samples were suspended in PBS buffer, driedon a glass window, to obtain a homogeneous film, and 500 – 1000 micron sample pressed using diamond compression cell. Spectra were recorded using Hyperion 2000 with15x IR objective; the analysed sample area was 100x100 microns. This approach allows to analyse significantly lower amount of sample compared to that of HTS-XT. For example, to gain a good quality spectrum using HTS-XT are required ~200000 cells whereas ~ 2000 cells using a diamond compression cell. FT-IR spectra of cells and EV recorded by HTS-XT or Hyperion correspondingly were qualitatively equal with similar absorbance. This study showed that compression cell is a valuable tool for studies of micro-biosamples by FT-IR microspectroscopy. Acknowledgment: This study was supported by the LCS joint project “Cancer-derived exosomes – a source of novel biomarkers and therapeutic targets for gastrointestinal cancers”

Poster Session

Deciphering Staphylococcus aureus Surface Glycostructures by FTIR Spectroscopy

T. Grunert1), S. Johler2), G. Xia3), F. Buzzola4), M. Ehling-Schulz1)

1) Functional Microbiology, Institute of Microbiology, University of Veterinary

Medicine, Vienna, Austria; 2) Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zürich,

Switzerland; 3) Institute of Inflammation and Repair, Faculty of Medical and Human Sciences,

University of Manchester, Manchester, United Kingdom; 4) Departamento de Microbiologia, Parasitologia e Imunologia, Facultad de Medicina,

Universidad de Buenos Aires, Buenos Aires, Argentina.

Staphylococcus aureus is an opportunistic pathogen that asymptomatically colonizes their hosts, but can sometimes cause serious acute and chronic infections in human as well as in animals. S. aureus surface glycopolymers, which includes capsular polysaccharide, cell wall teichoic acid and β(1-6)-N-acetylglucosamine (PNAG), play an important role during infection and some of them are interesting targets for vaccination. Recently, we could show that capsular polysaccharide expressing and non-expressing strains can be discriminated by metabolic fingerprinting by Fourier-transform infrared (FTIR) spectroscopy [1]. Furthermore, chemometrics-assisted fingerprinting of surface polysaccharides offers high-resolution subtyping of intact S. aureus strains [2] and can be used to differentiate modifications of the glycosylic pattern of S. aureus cell wall teichoic acid. Practical applications will be presented including the monitoring of general changes in S. aureus glycostructural composition due to gene knockouts and different treatments and of S. aureus intramammary infection (mastitis) in dairy cattle at herd level [3, 4]. In summary, metabolic fingerprinting by FTIR spectroscopy is a promising tool for differentiation and characterization of S. aureus strains based on their surface glycopolymer composition, thereby providing novel insights into staphylococcal pathogenesis. References [1] T. Grunert, M. Wenning, M. S. Barbagelata, M. Fricker, D. O. Sordelli, F. R. Buzzola, M. Ehling-Schulz, J

ClinMicrobiol. 51, 2261-6 (2013). [2] S. Johler, R. Stephan, D. Althaus, M. Ehling-Schulz, T. Grunert, SystApplMicrobiol. 39, 189-94 (2016). [3] C. Dotto, A. L. Serrat, N. Cattelan, M. S. Barbagelata, O. M. Yantorno, D. O. Sordelli, M. Ehling-Schulz,

T. Grunert, F. Buzzola, “The active component of aspirin, salicylic acid, promotes Staphylococcus aureus biofilm formation in a PIA-dependent manner”, Front. Microbiol. 23;8:4 (2017).

[4] J. Kümmel, B. Stessl, M. Gonano, G. Walcher, O. Bereuter, M. Fricker, T. Grunert, M. Wagner, M. Ehling-Schulz, “Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese”, Front Microbiol. 13;7:1603 (2016).

Poster Session

Modified Protein Complexes for Non-Invasive Molecular Control

Carolin Hartmann1, Susanne Pettinger2, Chris Massner2, Martin Elsner1, Reinhard Niessner1, Gil Westmeyer2, Natalia P. Ivleva1

1Technical University of Munich, Chair of Analytical Chemistry and Water Chemistry,

Marchioninistraße 17, Munich 2Technical University of Munich, Institute for Biological and Medical Imaging,

Ingolstädter Landstraße 1, Oberschleißheim

The possibility of an application of the iron storage protein Ferritin has gained great interest in various biological and medical fields as well as in nanotechnology. Ferritin is an universal intracellular iron storage protein of spherical shape with an outer and inner diameter of 12 and 8 nm, respectively. It possesses an iron core inside the cavity [1]. This cavity acts like a reaction chamber and is well-suited for the natural formation and storage of nano-sized particles through biomimetic mineralization. Furthermore, modified Ferritin called Magnetoferritin with an iron core that is loaded with the magnetic iron oxides magnetite and/or maghemite, would represent a promising substitute of synthesized magnetic nanoparticles for applications via magnetic fields. Hence, a non-destructive imaging inside living organism or manipulation of cells via magnetic fields would be possible. The usage of Magnetoferritin may also facilitate various applications of medical diagnostic methods such as hyperthermia or as contrast agents in MRI [2]. However, the knowledge of the exact native structure is not yet established, even though it would be essential for an understanding of the mechanism of the processes inside the iron core. Then, it would be possible either to modify and tailor Ferritin individually, for instance into Magnetoferritin, or to differentiate accurately real cell samples loaded with iron containing nanoparticles. Raman Microspectroscopy (RM) has become a versatile tool for investigations of proteins and cells in the past decades [3-5] and would be a promising approach for a non-destructive evaluation and monitoring of the structure and chemical composition of the core inside the protein. Based on the inelastic scattering of light, RM provides spectra which are unique to each compound and structure. Since water is a weak Raman scatterer and does not interfere with the measurements, RM is highly suitable for the analysis of biological samples embedded in their natural matrix. Due to this advantage minimal or no sample preparation is required and analyses can be carried out at room temperature. We investigate different iron storage proteins and characterize their iron core by means of RM. Further applications of RMfor the analysis of modified protein complexeshold potential to help in several studies, for instance for an understanding of the origin and progress of neurological diseases (e.g. Alzheimer´s disease). References [1] E. C. Theil, Inorg Chem. 52, 12223-12233 (2013).

[2] V. C. Jordan, M. R. Caplan, K. M. Bennett, Magnetic Resonance in Medicine 64, 1260-1266 (2010). [3] L. Ashton, V. L. Brewster, E. Correa, R. Goodacre, Analyst 142, 808-814 (2017). [4] R. Tuma,J Raman Spectrosc. 36(4), 307-319 (2005). [5] N. P. Ivleva, M. Wagner, H. Horn, R. Niessner, C. Haisch, J Biophoton. 3, 548-546 (2010).

Poster Session

Surface Enhanced Hyper Raman Spectroscopy for Bioapplications

Zsuzsanna Heiner, Fani Madzharova, Vesna Živanović, Marina Gühlke, Janina Kneipp

Humboldt Universität zu Berlin, 12489 Berlin, Germany

Surface enhanced hyper-Raman scattering (SEHRS) isthe basis of promising micro-spectroscopic applications, because it allows to collect structural and chemical information with high electromagnetic enhancement coming from plasmonic nanomaterials [1, 2]. Depending on the molecular symmetry, SEHRS may probe IR active modes or silent modes, which are seen neither in Raman nor in IR spectra [2, 3]. In addition, because the biological cells and tissues are transparent in the near infrared range, two-photon microscopy can offer advantages due to excitation at lower photon energy, deeper penetration, and better spatial resolution, while the detection stays in the visible spectral range [3]. While the applications of noble nanomaterials in the fields of biosensing, drug delivery, and therapeutics are spreading, many interactions between complex biomolecules and nanoparticles are not well understood. Here, we report the non-resonant SEHRS spectra of amino acids and antidepressant drugs and discuss their interactions with noble metal nanoparticles.Furthermore, we employed SEHRS based hyperspectral imaging for determining the location and spatial distribution of SEHRS labels inside live macrophages and for mapping physiological parameters (e.g., pH) in the endosomal system of the cells.

SEHRS spectra of tryptophan, histidine, phenylalanine, and tyrosine weremeasured and analyzed under small variations in concentration and surface environment. We found that the spectral differences are coming from the change in the orientation of the molecules on the nanostructure. In another set of experiments, the SEHRS spectra of tricyclic antidepressant molecules on silver and gold nanostructures were obtained. The comprehensive analysis of vibrational spectra clearly shows that the interaction of the molecules with the plasmonic nanoparticles strongly differs in the case of gold and silver surfaces,and that the characteristic bands of this interaction do not vary muchin acidic microenvironment.

As the first demonstration of SEHRS-based imaging, we show that in mapping mode, SEHRS fingerprints can be collected from live animal cells after incubation with SEHRS labels, that is, nanoparticles tagged with different molecules. Analyzing the data sets, chemical and pH maps can be determined. Applying multivariate signal processing techniques (e.g., principal component analysis, PCA) to SEHRS spectra, the distributions of structurally very similar reporter molecules can be mapped and distinguished.

The results of our experiments show high potential of this spectroscopic method in applications benefitting from its unprecedented sensitivity with respect to molecular structure and interaction.

Acknowledgement: Financial Support by ERC Starting Grant no.259432 (MULTIBIOPHOT) to J. K. and by DFG (GSC 1013 SALSA) to Z. H. and V. Ž. is gratefully acknowledged. F. M. acknowledges funding by a Chemiefonds Fellowship (FCI). References [1] J. Kneipp, H. Kneipp, K. Kneipp, PNAS 103, 17149-17153 (2006). [2] F. Madharova, Z. Heiner, J. Kneipp, J. Phys. Chem. C 121, 1235-1242 (2017). [3] Z. Heiner, M. Gühlke, V. Živanović, F. Madzharova, J. Kneipp, Nanoscale 9, 8024-8032 (2017).

Poster Session

Silver Halide Fibers and other Materials Relevant for Attenuated Total Reflection and Transmission Infrared Spectroscopy: Biocompatibility and

Toxicity Testing Using Fibroblast Cells

Sven Delbeck, Julia Hartmann, Nina Kumpf, Eva Eisenbarth, H. Michael Heise

Fachbereich Informatik und Naturwissenschaften, Fachhochschule Südwestfalen, Frauenstuhlweg 31, 58644 Iserlohn, Germany

Infrared spectroscopy is an important analytical method for in-vivo monitoring of different analytes in body-fluids. Recently, silver halide fibers have attracted much interest for implanting sensing devices for blood glucose using transmission spectroscopy [1]. With especially flattened fibers a large signal enhancement could be achieved using the attenuated total reflection (ATR) technique [2]. Therefore, the biocompatibility and toxicity towards in-vivo applications of silver halide fibers has to be confirmed.

Based on DIN EN ISO 10993-5:2009, the impact of flattened silver halide materials of different stoichiometric composition AgClxBr1-x (0<x<1) on fibroblast cells was compared with that from other interesting materials for infrared attenuated total reflection and transmission spectroscopy, such as calcium fluoride or various IR-transparent polymer foils (polyethylene, teflon). These cells (species: murine; cell line NIH-3T3; DSMZ no. ACC 59) usually form an adherent monolayer under specific growth conditions, e.g., with usage of Dulbecco’s modified eagle culture medium (90 % DMEM with 10 % of heat inactivated fetal bovine serum) and an incubation temperature at 37 °C. Cell-biological and infrared spectroscopic methods were applied for monitoring the influence of the different materials on the fibroblast cells for several days. Spectra of wet and air-dried cell layers were recorded in transmission and ATR mode for spectral comparisons after cell exposure. Further investigations were carried out for a live monitoring of cell sedimentation testing using the infrared attenuated reflection technique with flattened silver halide fibers. Spectral temporal changes in the biochemical composition of the fibroblast cells have been detected by using difference spectroscopy. For illustrating the proliferation process and the kinetics of growth, special dyeing methods were applied. In the presented work, the biocompatibility and toxicity characteristics of relevant materials have been highlighted from different scientific views and compared to each other. References [1] C. Vrancić, N. Kroger, N. Gretz, S. Neudecker, A. Pucci, W. Petrich, Anal. Chem. 86, 10511−10514 (2014). [2] Y. Raichlin, D. Avisar, L. Gerber, A. Katzir, Vibrational Spectroscopy 73, 67–72 (2014).

Poster Session

Developing a Rapid Screening Mid-IR Imaging Method for Diagnosis of Oesophageal Cancer

H. Sheridan1, M. Hermes2, N. Sheperd3, and N. Stone2

1Natural Sciences, College of Engineering, Mathematics and Physical Sciences, University of

Exeter, EX4 4QL 2Biomedical Physics, School of Physics and Astronomy, University of Exeter, EX4 4QL

3Gloucestershire Hospitals, NHS Foundation Trust, Gloucester, GL1 3NN

Barratt’s oesophagus (BO) is a condition wherein Gastro-oesophageal reflux disease (GERD) and acid reflux, colloquially referred to as heartburn, causes the oesophageal lining to replace its normal stratified squamous epithelium with simple columnar epithelium. This abnormal change of cells (metaplasia) is the cause of the increased likelihood of degeneration into dysplasia or adenocarcinoma. Therefore patients who present with BO are regularly monitored to check for progression of disease, in the form of an endoscopy to obtain biopsies for analysis by a pathologist. Subsequent to the collection of biopsies, samples are cut and stained with haematoxylin and eosin dyes before manual inspection by a pathologist. This procedure is time consuming and produces large volumes of biopsies for manual evaluation by a trained pathologist. This procedure could be replaced by hyperspectral infrared (IR) imaging techniques in the future [1].This work aims to optimise the process ofcomputing a digital stain using FT-IR data from unstained tissues by determining ideal imaging parametersto develop an imaging routine fora future, larger scale study. Data was collected from a paraffin embedded tissue micro array (TMA) containing biopsies of different pathologies, using an Agilent Carry 670 FT-IR microscope with 5.5 µm effective pixels size. Both percentage of beam attenuation and scan number were considered as parameters; with ideal attenuation determined by signal to noise ratio and optimum scan number ascertained using K means clustering. Ideal parameters were chosen as 25% laser attenuation and 16 scans, which were then used to image each biopsy of the TMA (acquisition time approx. 15 minutes per biopsy) in order to create digital stains and classification models for diagnostic purpose of the samples. Acknowledgement: The Authors thank the Association of British Spectroscopists and The European Union within the scope of the Horizon 2020 program [H2020-MSCA-ITN-2014- 642661] and the ABS Trust for funding this work. References [1] O. J. Old, G. R. Lloyd, J. Nallala, M. Isabelle, L. M. Almond, N. A. Shepherd, C. A. Kendall, A. C. Shore,

H. Barr and N. Stone “Rapid infrared mapping for highly accurate automated histology in Barrett's oesophagus” Analyst, in Press. (2017).

Poster Session

Detection of the Cellular Uptake and Localization of PhotoCORMs by Means of FT-IR Micro-spectroscopic Imaging

Patrick Hoffmanna,b, Ralf Medec, Marcel Dahmsb,d, Matthias Westerhausenc,

Jürgen Poppb,d,e, Ute Neugebauera,b,d,e

aCenter for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, D-07747 Jena, Germany

bLeibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, D-07745 Jena, Germany cInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena,

Humboldstraße 8, D-07743 Jena, Germany dInfectoGnosticsResearch Campus Jena, Center for Applied Research, Philosophenweg 7,

D-07743 Jena, Germany eInstitute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University

Jena, Helmholtzweg 4, D-07743 Jena, Germany

Over the last decades, the activity of endogenously produced carbon monoxide (CO) in a wide range of biological processes has been reported. Elucidating signaling pathways of CO and exploring the effect and therapeutic potential in human medicine is of recent interest. However, gaseous CO well-known for its inherent toxicity, is difficult to administer in an accurate quantity and at a specific location within the human body. Therefore, CO-releasing molecules (CORMs) are developed as CO delivery systems liberating the gas in a controlled manner by activation viaa stimulus [1]. The distinct intrinsic spectroscopic properties make CORMs well suited for studying their cellular uptake by Fourier transform infrared (FT-IR) micro-spectroscopy, since the carbonyl vibrational bands appear in a region usually silent for signals of biomolecules (1800 to 2200 cm-1, Fig. 1a). Furthermore, hyperspectral imaging using mid-IR light allows non-destructive investigation of mammalian cells treated with novel UV-Vis light sensitive photoCORMs. Experiments were performed on an Agilent Cary 670 FT-IR spectrometer with an Agilent Cary 620 FT-IR microscope equipped with a 25× objective and 64 × 64 FPA detector. Recorded data were evaluated and cellular CORM distribution visualized (Fig. 1b) usingthe programming environment R by applying the N-FINDR algorithm [2]. Detection of the cellular uptake ofnovel photoCORMs was successfully demonstrated by rapid and non-destructive imaging via FT-IR micro-spectroscopy.That result pave the way for future investigation of the effect of CO against human cell lines by application of innovativewater-soluble and non-toxic photoCORMs. Acknowledgments: Financial support by the DFG for research unit FOR 1738 (WP7 & 8) and by the BMBF for the Integrated Research and Treatment Center CSCC (FKZ 01EO1502) is highly acknowledged.Further thanks go to C. Krafft for providing the FT-IR instrumentation and J. Rüger for help with the data handling (both Leibniz IPHT). References [1] S. H. Heinemann, T. Hoshi, M. Westerhausen, A. Schiller, Chem. Commun. 50, 3644–3660 (2014). [2] C. Beleites et al., “A New N-FINDR Algorithm and the unmixR Package for Spectral Unmixing”, in: FT-IR

Spectroscopy in Microbiological and Medical Diagnostics, RKI Berlin, Germany (2015).

1000 1500 2000 2500 3000 3500

1800 1850 1900 1950 2000 2050 2100

IR a

bsor

ban

ce

Wavenumber [cm-1]IR

abs

orb

ance

Wavenumber [cm-1]

ν (CO)

- CORM- Cell

Figure1.a) IR mean spectrum of cells after treatment with aqueous CORM solution evealing appearance of specific CO marker bands (Inset) and b) the regarding IR false color abundance plot of cell and CORM fractions.

a)

b)

    Poster Session  

PM-IRRAS and AFM Studies on Modified ssDNA Adsorbed on Gold

A. Jaworska, A. Jabłońska, B. Pałys, A. Kudelski

Faculty of Chemistry, University of Warsaw, Pasteur 1, 02-093 Warsaw, Poland Polarization modulation infrared reflection adsorption spectroscopy (PM-IRRAS) is a highly surface specific FT-IR method that is capable of detecting chemical compositions from interfacial films down to one molecule thin films [1]. The PM-IRRAS technology enables the measuring spectra of materials because of the differences in the reflection of p- and s-polarized light from interfaces – only p-component radiation interacts with the surface of sample [2]. The PM-IRRAS technique allows enhanced detection on substrates and measurements from the air-water interface. Changes in the PM-IRRAS signal intensity and position can be used to infer molecular absorption/desorption behaviour and kinetics, molecular packing, phase transitions, hydration, hydrogen bonding and different surface reactions in a thin film. Additionally in PM-IRRAS the properties of the polarized light can be used to determine the molecular orientation in a film [3]. Here we present the studies on ssDNA consisting of 20 molecules of adenine adsorbed on flat gold surface, also modified with thiol and anion groups at the 5’ ending. Atomic force microscopy images were measured to confirm the presence of ssDNA on the surface together with its distribution. The influence of the presence of complimentary ssDNA in the sample on the PM-IRRAS signal was tested due to investigate the hybridization process. References [1] T. Buffeteau et al., Applied Spectroscopy 45, 380-389 (1991). [2] Alkire, Kolb, Lipkowski, Ross (eds.): Diffraction and Spectroscopic Methods in Electrochemistry, Wiley-

Vch (2006). [3] R. Arnold et al., Langmuir 17, 4980-4989 (2001).

Figure 1. PM-IRRAS spectra of 5’-AAAAAAAAAAAAAAAAAAAA-3’(A20) molecule and itsthiol (A20-C6-SH) and amine (A20-C12-NH2) modifications.

Poster Session

AFM-IR Nanoscale Study of Cell Walls in Living Bacteria

Kamila Kochan1, David Perez – Guaita1, Julia Pissang1, Jhih-Hang Jiang2, Anton Peleg2,3, Philip Heraud1,2, Bayden Robert Wood1

1Centre for Biospectroscopy and School of Chemistry, Monash University, Clayton Campus,

3800, Victoria, Australia 2Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department

of Microbiology, Monash University, Clayton, Victoria 3800, Australia 3Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash

University, Melbourne, Victoria 3004, Australia *kamila.kochan@monash.edu,

The AFM-IR combines the advantages of two commonly used research techniques: Atomic Force Microscopy (AFM) and Infrared Spectroscopy (IR).1 The first allows one to measure the local physical properties of a sample (height or probe deflection) with nanoscale accuracy, whereas the latter enables the chemical characterization of a sample. Although IR spectroscopy can provide information on a molecular level, its major limitation is the wavelength diffraction limited spatial resolution restricting it to the study on a microscale rather than nanoscale. This limitation is overcome by coupling IR spectroscopy with AFM. The fundamental process occurring during the measurement via AFM-IR is still the infrared absorption, however, it is usually not measured in a direct way.1 The absorption of IR radiation results in a local increase of the temperature, which further leads to a force impulse causing the oscillation of the AFM probe. The thermal expansion phenomenon is directly dependent on the sample composition, but the strength of the signal depends also on the material properties.1 Therefore, the thermal expansion phenomenon as well as detailed capabilities of the technique (such as spatial resolution) are not precisely defined yet.

The ability to achieve nanoscale spatial resolution of IR result in rapidly increasing popularity of AFM-IR for the study of biological materials, opening up the possibility to investigate small single-celled organisms, such as bacteria..2 In our study we focused particularly on the cell envelope of bacteria, which differs significantly between Gram-positive and Gram-negative classes of bacteria. Gram-positive bacteria have a thick cell wall layer that covers the cytoplasmic membrane, whilst Gram-negative bacteria have outer- and inner- membranes with peptidoglycans in between. The cell wall structure of Gram-positive bacteria is of particular importance from a clinical point of view, as disruption of cell wall biosynthesis by antibiotics has been effective to treat Gram-positive bacterial infections.

In our study we were able to investigate several representatives of Gram-positive and Gram-negative bacteria in vivo and demonstrate the pronounced differences between their AFM-IR spectra, attributed to characteristic cell wall components (e.g. peptidoglycan, teichoic acid). We demonstrate – on the example of Escherichia coli – that even in the case of Gram-negative bacteria with a thin cell wall, it is possible to examine this structure via AFM-IR in a live, intact organism. In addition, we demonstrate for the first time a live Staphylococcus aureus cell during the formation of its septum prior to division probed with the AFM-IR, showing an increased intensity of bands identified earlier as related to peptidoglycan and teichoic acid in the newly formulating cell wall. References [1] A. Dazzi, C. B. Prater, Chemical Reviews (2016), DOI: 10.1021/acs.chemrev.6b00448 [2] A. Dazzi et al., Ultramicroscopy 108(7), 635 – 641 (2008).

Poster Session

Optofluidic Platform for Enhanced IR Microscopic Biosensing

C. Kratza, T. W. H. Oatesa, D. Janasekb, K. Hinrichsa

a Leibniz-Institut für Analytische Wissenschaften —ISAS— e.V., Schwarzschildstr. 8, 12489 Berlin, Germany

b Leibniz-Institut für Analytische Wissenschaften —ISAS— e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany

An optofluidic platform for in situ enhanced IR microscopic biosensing is presented enabling structural and chemical analysis of biomolecules in nanoliter liquid samples. The platform combines enhancement substrates and microfluidic chips of arbitrary material and is designed for applications in conventional IR microscopes [1]. Potential applications for the platform are lab-on-chip, organ-on-chip, cell analytics, bio-analytics or diagnostics.

In situ IR spectroscopy enables label free and non-destructive molecular identification and can provide detailed information on interactions and reactions e.g. changes in protein folding or receptor-ligand interactions. The technique has proven its application potential for in situ studies of e.g. technologically relevant thin films [2], bio-functional surfaces [3] and cells [4] in liquid environments.

The presented optofluidic platform circumvents the challenge of strong IR absorption of common polymeric materials used for microfluidic chips employing a single-reflection geometry under non-ATR (attenuated total internal reflection) conditions. This development allows for the usage of commercially available chips facilitating the application of the method. Utilization of optimized enhancement substrates of metal island films [5] offers a signal enhancement by a factor of 10-100 mediated by the effect of surface enhanced infrared absorption (SEIRA) [6].

Submonolayer sensitivity of the developed platform is demonstrated by studying the formation of 1.2 nm thin monolayers of the tripeptide glutathione (GSH) in an aqueous environment. The potential to study interactions and reactions at the solid–liquid interface is shown by monitoring chemical changes in the monolayer as response to changes in environmental pH. Time-resolved (2.5 min) measurements of GSH monolayer formation show that dynamic processes can be monitored in situ. The determined detection limit of 0.03 nmol/cm2 emphasize the potential of the platform in biosensing applications as well as in the study of dynamic processes e.g. enzymatic reactions, receptor–ligand interactions or conformational changes of molecules due to environmental stimuli with sub-monolayer sensitivity under in situ conditions. References [1] C. Kratz, A. Furchner, T. W. H. Oates, D. Janasek and K. Hinrichs, submitted

[2] A. Kroning et al., ACS Appl. Mater. Interfaces 7, 12430-12439 (2015).

[3] P. R. Griffiths, J. Chalmers (eds), “Handbook of Vibrational Spectroscopy”, Wiley (2002).

[4] H. Y. N. Holman et al,. Analytical chemistry 81.20, 8564-8570 (2009). [5] C. Kratz, T. W. H. Oates, and K. Hinrichs, Thin Solid Films 617, 33-37 (2016). [6] M. Osawa, in S. Kawata (eds): “Near-Field Optics and Surface Plasmon Polaritons” p. 163-187, Springer

(2001).

Poster Session

Investigation of Therapeutic Effect of Palmitoleic Acid on Obesity Induced Type 2 Diabetes in Adipose Tissue by Fourier Transform Infrared

Microspectroscopy

Fatma Küçük Baloğlu1,2, Feride Severcan1,3

1Middle East Technical University, Department of Biological Science, Ankara, Turkey 2Giresun University, Department of Biology, Giresun, Turkey

3Kemerburgaz University, Department of Biophysics, İstanbul, Turkey

Fundamental investigations on obesity and obesity related type 2 diabetes which are common diseases nowadays are so important for therapeutic studies in future. In the light of this information, it is firstly aimed to investigate the structural and compositional alterations in adipose tissue caused by obesity related type 2 diabetes. Secondly, a preliminary study on the therapeutic effect of palmitoleic acid on these alterations was performed by investigating its potential of reversal effect. Obesity is especially characterized by adipocyte hypertrophy, the expansion of visceral and subcutaneous adipose tissue mass in the body, and alterations in cellular biology. The increase of the expansion of visceral (VAT) and subcutaneous (SCAT) adipose tissue mass is the main reason of obesity and mostly this process results in disturbed glucose and lipid metabolism. Since visceral and subcutaneous adipose tissues have a critical role in obesity related type 2 diabetes, we studied with these two types adipose tissues. In this project, we investigated the molecular alterations in the concentration and composition of obesity related type 2 diabetes in adipose tissue by Fourier Transform Infrared (FTIR) microspectroscopy. Afterwards, a preliminary study which gives direction to reveal the potential of palmitoleic acid as an anti-obesity and anti-diabetic drug is carried out by the same technique. The results of the current study revealed that obesity and type 2 diabetes caused some alterations in molecular composition in adipose tissue as an increased lipid/protein ratio, an increased carbonyl/lipid ratio and a decreased unsaturated/saturated lipid ratio. However, our preliminary studies indicated that the palmitoleic acid have areversal effect on these destructive alterations of obesity and type 2 diabetes. Therapeutic effect of palmitoleic acid on these destructive alterations of obesity and type 2 diabetes were valid for both SCAT and VAT significantly. This study could be the basis of the further studies on treatment of obesity related type 2 diabetes mechanism.

    Poster Session

Application of AFM-IR to Study Human Lenses and Chromosomes

W. M. Kwiatek1, A. Borkowska1, E. Lipiec1, E. Pięta1, N. Piergies1, and C. Paluszkiewicz1

1Institute of Nuclear Physics Polish Academy of Sciences, PL-31342 Krakow, Poland

The combination of AFM and IR techniques (AFM-IR), provides both IR - spectra on nanoscale level and topography of the samples which is given by the AFM [1]. NanoIR2 system at the Institute of Nuclear Physics PAN in Krakow, Poland enables the use of both OPO (Optical Parameter Laser) and QCL (Quantum Cascade Laser) lasers. Application of OPO and QCL lasers results in different features in the IR-spectra [2]. The presentation will describe two different applications of AFM-IR study. First example is human lens tissue. Cataract is a widespread disease, which leads to cloudy or misty vision related to the decrease of eye lens transparency [3].The use of RS, FTIR, and AFM-IR techniques make them attractive tools to characterize biological components. Fig. 1 presents the images of the healthy and cataractous human lenses at micro- and nano- scale levels along with the Amide I distribution maps. The obtained data indicate the influence of the disease development on the secondary structure of proteins and conformational changes of the amino acid residues. Figure 1. Microscope and AFM images of healthy (A and B) and cataractous lens (C and D) tissues, with corresponding Amide I intensity maps recorded by FTIR and AFM-IR, respectively. The second example is human chromosomes. Theyare composed of chromatin that is a mixture of nucleic acids (DNA) and proteins (histones) [4]. QCL and OPO lasers were applied to compare AFM-IR signal. The results show enormous difference in quality and quantity of measured bands. When QCL laser was used the 1720 cm-1, 1660 cm-1, 1550 cm-1, 1460 cm-1 and 1230 cm-1 bands were very well defined while in case of OPO laser the 1540cm-1 and 1460 cm-1bands weren’t detected.

Figure 2. AFM-IR maps and spectra of single chromosome: a- AFM topography of acentric single chromosome, b- deflection signal, c- AFM-IR distribution of νas (O- P-O) 1230 cm-1 band, IR Peak measured using OPO laser, d- AFM-IR distribution of 1230 cm-1 band measured using QCL laser, averaged spectrum measured with e- OPO laser and f- QCL laser.

Acknowledgment: This project has been supported by the National Science Centre Poland under decision no. DEC-2012/05/B/ST4/01150. This research was performed using equipment purchased in the frame of the project co-funded by the Małopolska Regional Operational Program Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project No. MRPO.05.01.00-12-0/1513.

References [1] C. Paluszkiewicz, N. Piergies, P. Chaniecki, et al., J. Pharmaceut. Biomed. 139, 125-132 (2017). [2] A. Dazzi, C. B. Prater, Q. Hu, D. B. Chase, J. F. Rabolt, and C. Marcott, Appl. Spectr. 66, 1365 (2012). [3] Z. Zhuang, M. Zhu, Y. Huang et al., Appl. Phys. Lett. 101, 173701-173701 (2012). [4] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, “Chromosomal DNA and Its Packaging

in the Chromatin Fiber”, in Molecular biology of the cell, 4th edition, (2002), 1

A B

C D

    Poster Session  

Identification of Pollen Grains in Mixtures Using Hyperspectral MALDI-TOF MS Imaging

F. Lauer1,2, S. Diehn1,2, S. Weidner1,2, J. Kneipp1,2

1BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Str. 11,

Berlin 2Humboldt-Universitätzu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, Berlin

Anemophilous plants produce pollen grains, which promote allergies. Therefore, pollen are monitored to provide a national information network. Their conventional identification and differentiation is performed by time-consuming microscopic examinations based on the genus-specific pollen morphology. A variety of spectroscopic and spectrometric approaches have been proposed to develop a fast and reliable pollen identification using specific molecular information [1, 2]. Amongst them, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a high potential for the successful investigation of such complex biological samples [2]. Specifically, it was illustrated that MALDI-MS imaging provides a powerful tool to identify pollen grains in pollen mixtures on the basis of ion intensity plots [3]. More recently, the evaluationof the obtained peak patterns from pollen mass spectra with multivariate statistics enables a consistent and rapid identification of the taxonomic relationships [4]. A novel application using conductive tape on the MALDI target simplifies sample preparation and enhanced the quality of the mass spectra [5]. This led to a comprehensive analysis of the MS patterns, which is important when identifying pollen grains from different plant species in mixtures.

Here, we present further developments in MALDI-MS imaging of mixtures of pollen from different plant species. By combining conductive tape sample preparation with MALDI MSI and chemometric analysis, first promising results were obtained. In addition, we discuss the ability of partial least square regression (PLS-R) to identify pollen species based on independent reference spectra and present first results obtained with artificial pollen mixtures. These methods will be used in future online identification of pollen species innatural pollen mixtures. Acknowledgements: F. L. and S. D. would like to thank Dr. Stephan Seifert for his work on the analysis tool and for fruitful discussions. J. K. acknowledges funding by ERC grant 259432 MULTIBIOPHOT. References [1] F. Schulte, J. Lingott, U. Panne, J. Kneipp, Anal. Chem. 80, 9551-9556 (2008). [2] B. Krause, S. Seifert, U. Panne, J. Kneipp, S. M. Weidner, Rapid Commun. Mass Spectrom. 26, 1032-1038

(2012). [3] S. M. Weidner, R. D. Schultze, B. Enthaler, Rapid Commun. MassSpectrom. 27, 896-903 (2013). [4] S. Seifert, S. M. Weidner, U. Panne, J. Kneipp, Rapid Commun. Mass Spectrom. 29, 1145-1154 (2015). [5] F. Lauer, S. Seifert, J. Kneipp, S. M. Weidner, Int. J. Mol. Sci. 18, 543 (2017).

Poster Session

Interaction of Magnetic Nanorods Coated by Dopamine with Anionic Liposomes as Revealed by FTIR Spectroscopy

I. M. Le-Deygen, E. D. Kutsenok, M. V. Efremova, P. G. Rudakovskaya,

A. G. Majouga, Y. I. Golovin, E. V. Kudryashova, A. V. Kabanov, N. L. Klyachko

Lomonosov Moscow State University, 119991 Russia, Moscow, Leninskie gory 1, 3

i.m.deygen@gmail.com

Liposomes are almost ideal carriers for delivery of both hydrophobic and hydrophilic drugs and genes. Nowadays one of the key problems is the cargo loading and controlled release. It is accepted based on the results of recent studies that radio frequency, low frequency, and extremely low frequency magnetic fields penetrate easily through the body providing great promise for internal therapy. However, the molecular mechanism of effects observed in magnetoliposomes is not entirely understood yet. Local bilayer disordering, which presumably can be achieved by oscillation of membrane-bound magnetic nanoparticles, may also result in the liposome cargo release.

A direct evidence of liposomal membrane loosening by alternating extremely low frequency magnetic field (ELF MF)-mediated nanorods oscillation is presented in this work and physical model of this process is suggested. We have studied a system based on anionic liposomes containing 20 wt % of anionic lipid cardiolipin (CL) and 80 wt % of dipalmitoylphosphatidylcholine (DPPC) and magnetic nanorods coated with positively charged dopamine (f-MNPs). f-MNPs and liposomes were characterized by DLS and NTA methods to estimate hydrodynamic radius rHD and concentration of particles. The process of membrane loosening under magnetic field was demonstrated by a novel technique based on Attenuated Total Reflection Fourier Transform IR spectroscopy (ATR-FTIR) about 50% of hydrophobic chains became highly mobile under the action of magnetic field. These results are corroborated by classic label-based fluorescence spectroscopy. Using sodium chloride as an example we show that the phenomenon of membrane fluidity increase is accompanied by the increase of membrane permeability causing the release of liposome cargo. Acknowledgement: This work is supported by RSF 14-13-00731 and RFBR 17-54-33027grants.

The roumultipleand labbetweenthe usephotodyresults (MAL)

glycogelesions, revealedthe postdespite

Figure performlesions the firscompardata mabetter ddiagona

Infrared Premalig

Cassio Aa UnAv.

bUniversit

utine molecue assays/sta

bel-free anan biomolecuefulness ofynamic therobtained byas photosen

en content. Pindicating

d an increast-PDT lesiontheir morph

1. Results omed between u

against healthst two princiison are depi

atrix and the lodiscriminated al.

d Spectroscgnant Skin

Aparecido

iversity of S. Prof. Lineuty of São Pa

ular techniqaining to stualytical toolules and nof FTIR sprapy in dyspy the treatmnsitizer prod

PDT-treatedg the presense in the amns, indicatinhological sim

obtained for untreated andhy skin. The ipal componected above thoading plots athe data are d

copy Deten Lesions

o Limaa, L

São Paulo, Nu Prestes 22aulo, Schoo05508-000

ques used toudy tissue-sl, which is ot only on aectroscopy

plastic cutanment using drugs.

d tissue exhnce of remmount of α-ng that treatmilarities.

pairwise cod PDT-treatedscore plots obents for eachhe main diagoassociated to tdepicted below

ermining t Submitte

Luciana Co

Nuclear and242, 05508-l of Dentist

0, Sao Paulo

o study cansamples. Oable to stu

a single oneto detect

neous lesion5-aminolev

AcOInhb

hibited similmaining lesi-helix and βted and non

omparisonsd cutaneousbtained forh pairwiseonal of thethe PC thatw the main

the Biocheed to Photo

orreab, Den

d Energy In-000, Sao Ptry, Av. Proo-SP, Brazil

ncer cells arn the otherudy the moe. The pres

the biochns, as well avulinic acid

Analysis tchanges proOverexpresII vibrationnon-treatedhealthy skinband intenlar histologiions after tβ-sheet secon-treated tiss

emical Chodynamic

nise Maria

stitute Reseaulo-SP, Brf. Lineu Prel

re time-conr hand, FTIolecular intent study ahemical chas to interrod (ALA) an

Swiss micchemical protocol aicutaneous submitted of ALA/Mwere collsections ofthe range ousing a infrared Attenuated(ATR) samspectra swere suPrincipal

to assess omoted by tssion of prons) was evd and posn, as well a

nsity of coical characttreatment. Fondary strucsue are bioc

Poster S

hanges in c Therapy

a Zezella

earch, razil estes 2227,

suming andIR provideseractions o

aims to demhanges indugate the thend its meth

ce underwecarcino

iming to inlesions an

to a singleMAL-PDT. lected fromf 5 µm thicof 4000 – 4Fourier trspectromet

d Total Refmpling mosecond derubmitted

Comthe bioc

the treatmenoteins (Amiidenced cost-PDT tisas a decreasollagen fibteristics to uFTIR spectcture of prochemically d

Session

d require s a rapid occurring monstrate uced by erapeutic hyl ester

ent to a ogenesis duce the nd were e session

Spectra m tissue ckness in 400 cm−1 ransform ter on flectance ode. The rivatives to the

mponents chemical nt. de I and

omparing sues to se in the

bres and untreated troscopy

oteins on different

Poster Session

Confocal Raman Imaging Integrated with Non-Negative Matrix Factorization Analysis on Spatiotemporal Distribution of Major Components in Biofilm

Xiao-Yang Liu1, 2, Shu-Xia Guo1, 2, Thomas Bocklitz1, 2, Petra Rösch1, 2,

Jürgen Popp1, 2, 3

1Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University,

Helmholtzweg 4, D-07743 Jena, Germany 2 InfectoGnostics Research Campus Jena, Philosophenweg 7, D-07743 Jena, Germany

3 Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena, Germany

Biofilms represent the predominant form of microbial life on our planet. The formation of biofilm is intrinsically complex, in which the microbial cells adhere to the surfaces and subsequently the extracellular polymeric substances (EPS) accumulate and undergo spatiotemporal changes [1]. Owing to the important features such as being rapid, noninvasive and label-free, Raman imaging becomes a powerful tool to study bacteria and chemical compositions of biofilm [2]. Although univariate data analysis has been commonly employed to construct Raman images, the severely overlapped spectral information could not be avoided [3]. Here, confocal Raman imaging combined with non-negative matrix factorization (NMF) analysis was applied to investigate the development of Escherichia coli biofilm. The biofilm was cultured statically at different ages and then measured by a Raman microscope. Our study provides direct observation of the biofilm formation, starting at the bacterial aggregation until biofilm maturation. The spectroscopic signature varied in plane and depth, showing the chemical heterogeneity of the biofilm. The major components of the biofilm, i.e. bacteria, lipid, protein, polysaccharides and lipopolysaccharide (LPS), could be distinguished and the spatiotemporal distribution is presented. The results show that the amount of EPS increased with time. Compared to bacteria, the EPS became the dominating feature of aged colonies. In conclusion, NMF analysis integrated with confocal Raman mapping could provide a comprehensive insight into the spatiotemporal distribution of various components of biofilm during the formation process, which may help to investigate the effects of various additives and environmental factors on biofilm growth. Acknowledgement: X. L. and S. G. acknowledge the funding from the China Scholarship Council (CSC). References [1] R. Janissen, D. M. Murillo, B. Niza, P. K.Sahoo, M. M. Nobrega, C. L. Cesar, M. L. A. Temperini, H. F.

Carvalho, A. A. de Souza, M. A.Cotta, Sci. Rep. 5, 9856 (2015). [2] B. Lorenz, C. Wichmann C, S. Stöckel, P. Rösch, J. Popp, Trends Microbiol. 25, 413-424 (2017).

[3] R. N. Masyuko, E. J.Lanni, C. M. Driscoll, J. D. Shrout, J. V. Sweedler, P. W.Bohn, Analyst 139, 5700-5708 (2014).

    Poster Session  

Preparation of Blood Samples for Raman Microspectroscopy on Single Bacteria Cells

Björn Lorenz1,2, Petra Rösch1,2, Jürgen Popp1,2,3

1Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University,

Helmholtzweg 4, D-07743 Jena, Germany 2InfectoGnostics Research Campus Jena, Philosophenweg 7, D-07743 Jena, Germany

3Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, D-07745 Jena, Germany In case of bacteremia, a patient has a risk to develop sepsis [1]. Sepsis belongs to the world leading causes of death due to the short time window before patients’ decease [2, 3]. In contrast, the gold standard blood culturerequires several hours up to days for cultivation [4]. However, timely description of the right antibiotic is vital considering the today’s prevalence of resistance among pathogens [2]. Consequently, the bacteria species has to be identified, although there might be less than10 bacteria presentin contrast to over 5·109 blood cells within 1 ml of blood [5, 6]. Within the need for better cultureless methods, Raman microspectroscopy has already proven to be fast with excellent bacterial identification capabilities for other scientific questions [7, 8]. The prerequisite to apply Raman microspectroscopy is to isolate nearly unaffected pathogens with a minimum of matrix [7]. Here, we present an isolation strategy to obtain single bacteria cells from blood tailored for Raman microspectroscopy by utilizing a lysis step and a hemoglobin removal step. Further, we prove the suitability of the isolated bacteria for Raman microspectroscopic identification. Acknowledgement: Funding of research project InterSept (13N13852) from the Federal Ministry of Education and Research (BMBF), Germany, is gratefully acknowledged. References [1] J. L. Vincent, Y. Sakr, C. L. Sprung et al., Crit Care Med. 34, 344-53 (2006). [2] A. Kumar, D. Roberts, K. E. Wood et al., Crit Care Med. 34, 1589-96 (2006). [3] L. Epstein, R. Dantes, S. Magill et al., Mmwr-Morbidity and Mortality Weekly Report 65, 342-345 (2016). [4] J. M. Ruiz-Giardin, R. M. Martin-Diaz, J. Jaqueti-Aroca et al., Int J Infect Dis. 41, 6-10 (2015). [5] J. A. Kellogg, J. P. Manzella and D. A. Bankert, J Clin Microbiol. 38, 2181-5 (2000). [6] B. George-Gay and K. Parker, J Perianesth Nurs. 18, 96-114; quiz 115-7 (2003). [7] S. Kloss, B. Lorenz, S. Dees et al., Anal Bioanal Chem. 407, 8333-41 (2015). [8] D. Kusic, B. Kampe, P. Rosch et al., Water Res. 48, 179-89 (2014).

 

Keywor

With thhigher eantibiotcompoutarget bunderstathe mosspatial rAFM-IRspatial rsmaller developAFM-IRand spatWe willpolymernew insmethodsfollow t

From leftimage- IR Referen[1] A. Da

resolu2390

[2] F. Ludetect

Label-F

J. Mathu

rds: IR nano

he increase efficiency atics can infunds with nbacteria witand the strust effectiveresolution. R microscopresolution o

than the fpments focuR or RE-AFtial resolutil discusstheric nanoparsight abouts. In the sethe localizat

t to right: PrinR nanospectro

nces azzi, R. Prazerution with an a(2005). u, M. Jin, Mtion”, Nat. Ph

Free Imag

urin*, E. P

* Laboratoi15 Rue G

e

ospectrosco

of antibiotand better sfluence the nanoparticleth higher doucture and i studies sh

py is a wellof an AFM mfar field difusing on resFM-IR) by ion up to 10e possibilitierticles. In tht the structecond part, tion of nano

nciple of the teoscopy of Nps

res, F. Glotin,atomic force m

M. A. Belkin, oton. 8, 307–3

ging to Chby IR N

Pancani, A

ire de ChimGeorges Clee-mail: jerem

opy, label fre

tic resistancselectivity s

general coesas deliveroses of antnternal chem

hould involv

l-establishedmicroscopeffraction limsonant excia tunable p

0 nm [2]. es to apply Rhe first part,ture of polywe will de

oparticles in

echnique- Polwithin macro

J. M. Ortega,microscope tip

“Tip-enhanc312 (2014).

haracterizNanospect

A. Deniset-

mie Physiqueemenceau, 9mie.mathuri

ee imaging,

ce significasolutions. Hondition of ry vehicles tibiotics. Fomistry of nve label-fre

d technique and a tunabmit by meaitation of thulsed sourc

RE-AFM-IR we will shymeric nan

emonstrate tnside fixed m

lymeric nanopophage: topogr

, “Local infrarp used as a ph

ed infrared n

e New Antroscopy

-Besseau,

e, Universit91405 Orsayin@u-psud.

, polymeric

ant effort isHowever, th

patients, thmay be a

or such devanoparticle/

ee techniqu

first demonble IR sourcasuring mohe AFM cance led to sig

R as a label ow how the

noparticles the use of macrophage

particle (NPs)raphic image

red microspechotothermal s

nanospectrosc

ntibiotics C

R. Gref, A

é Paris-Sudy - France fr

nanoparticl

s focused oe side effec

herefore usigood solut

velopments /antibiotic ses capable

nstrated in 2ces to reachlecular expntilever (Regnificant inc

free technie method cacompared RE-AFM-Ies at the sub

: theoretical sand chemical

ctroscopy withensor”, Opt. L

opy via mole

Poster S

Carriers

A. Dazzi

d,

le, macroph

n research cts of high ing first getion to speit is mand

systems.To of sub-mic

2005 combih optical respansion [1].esonance Ecrease in se

que for the an be used twithlow reR as a newbcellular sca

cheme and tomap.

h subwavelengLett.30, Issue

ecular expans

Session

hage

towards efficacy

eneration ecifically datory to

this end crometer

ining the solutions . Recent

Enhanced ensitivity

study of to reveal esolution w tool to ale.

pographic

gth spatial 18, 2388-

sion force

Poster Session

Development of Raman Platform for Single Cell Analysis

Abdullah S. Mondol1, Iwan W. Schie1, Jürgen Popp1,2

1Leibniz Institute of Photonic Technology Jena, Albert-Einstein-Straße 9, 07745 Jena, Germany

2Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Helmholtzweg 4, 07743 Jena, Germany

Raman spectroscopy provides a label-free molecular fingerprint of a sample and has been widely used to extract the biochemical composition of biological samples, both qualitatively and quantitatively. Because it provides a complete molecular fingerprint label-free, applications in single cell analysis [1] have been manifold, e.g. drug-cell interaction [2], identification of apoptotic and proliferate cells [2], toxicology [3], are just some of many applications [3]. While Raman spectroscopy cannot fully replace fluorescence microscopy, due to the lower acquisition speed, it can be a complimentary tool for standard research biology and clinical applications. Currently, however, Raman spectroscopy is only used in a research environment. The challenges for limited use of Raman spectroscopy arises due the complex data acquisition procedure for single cell Raman spectroscopy, which requires extensive human interaction to perform measurements as well as complex data post-processing, making the method less favorable. Moreover, the number of sampled cells is usually low, on the order of a few hundreds of cells, which results in low statistical significance. Here we present an approach to improve the data acquisition procedure for single cells Raman spectroscopy, by removing the human factor of the data acquisition. The proposed implementation allows the acquisition of a large number of cells rapidly, and an entire Raman experiment, can be performed without human intervention. This increases the efficiency for data acquisition and allows the performance of Raman experiments with no prior knowledge of the technology. References [1] I. Notingher, Sensors 7(8), 1343-1358 (2007). [2] I. Pence, A. M. Jansen, Chem Soc rev. 45(7), 1958-1979 (2016). [3] I. W. Schie, T. Huser, App. Spec. 67(8), 813-828 (2013).

    Poster Session  

Visualization of Pollutant Degrading Bacteria via Bioorthogonal Noncanonical Amino Acid Tagging Coupled to Surface-enhanced Raman Scattering

O. Morgaienko, N. P. Ivleva, M. Elsner

Technical University of Munich, Institute of Hydrochemistry, Chair of Analytical Chemistry

and Water Chemistry, Marchioninistr. 17, 81377 Munich, Germany Removal of organic micropollutants in water is crucial for drinking water quality as well as for maintaining aqueous ecosystems. Micropollutants are organic pollutants presented at extremely low concentrations with unknown impact on ecosystems. Conventional activated sludge systems employed in the most of wastewater treatment plants are typically not designed to remove micropollutants completely. Therefore, it is important and challenging to identify and characterize the microorganisms responsible for degradation of micropollutants during wastewater treatment [1]. Additionally, it is relevant to find out the microorganisms capable of maximal level degradation of organic pollutants ensuring sustained and cost efficient treatment approaches. Bioorthogonal noncanonical amino acid tagging (BONCAT) has been demonstrated as an effective tool to study individual cell response to external signals in situ [2]. Raman microspectroscopy (RM) allows nondestructive analysis of microorganisms at the single cell level. The problem of low sensitivity of RM can be overcome by the use of the surface-enhanced Raman scattering (SERS) effect [3]. Furthermore, by bringing together two of the most promising experimental approaches of different disciplines (BONCAT + SERS) reliable and detailed analysis of the bacterial substrate assimilation, efficient labelling, visualization and separation of cells without destroying or inhibiting bacteria can be achieved. An essential advantage of such an approach should be a single cell level scale of analysis. The aim of the study is to establish an approach based on the BONCAT technique that allows providing single cell level analysis and separation of the bacteria responsible for complete pollutant degradation. The proposed project sets out the protocol for coupling BONCAT and SERS for further studies of bacterial consortia under different conditions in various areas, including biodegradation. To this end, aims of the study are: (a) screening of the suitable bacteria for model experiments for the BONCAT-SERS research; (b) incorporation of amino acid analogues (AAAs) that carry chemical tags amenable to click chemistry into bacteria; (c) preparation of silver and gold nanoparticles (NPs) for SERS analysis; (d) coupling of bacteria, which have incorporated tagged AAAs with NPs based on the click reaction; (e) SERS analysis and determination of a suitability of the junction of BONCAT and SERS as a combination of two methods; (f) establishment of BONCAT for separation of bacterial cells incorporated AAAs with magnetic nanocomposites for further investigations. Applied approaches are expected to shed light on the ecophysiological diversity in the bacterial community and improve the understanding of microbial degradation of organic pollutants. References [1] C. Grandclement, I. Seyssiecq, A. Piram, P. et al., Water Res. 111, 297-317 (2017). [2] R. Hatzenpichler, S. A. Connon, D. Goudeau et al., Proc Natl Acad Sci USA 113, 4069-4078 (2016).

[3] N. P. Ivleva, P. Kubryk, R. Niessner, Anal. Bioanal. Chem. 409, 4353-4375 (2017).

Poster Session

Studies of Cancerous Tissues Composition Using FTIR and Raman Microspectroscopy Methods

C. Paluszkiewicz1, E. Pięta1, N. Piergies1, G. Lisowska2, M. Misiołek2,

W. Ścierski2, W. M. Kwiatek1

1Institute of Nuclear Physics Polish Academy of Sciences, PL-31342 Krakow, Poland 2Medical University of Silesia, Department of Otorhinolaryngology and Laryngological

Oncology in Zabrze, 41-800 Zabrze, Poland

Cancer is still the main reason of morbidity and mortality among people around the world [1]. According to the World Health Organization (WHO) in 2012 about 8.2 million people died due to the development of tumors. This number will increase by 70% within the next two years. The neoplasms arising among the head and neck region are the crucial problem in laryngological practice. These tumors are often diagnosed in salivary glands and paranasal sinuses [2]. These non-malignant types of cancer can often transform to pathogenic forms what promotes the development of malignant tumors [3].

Vibrational spectroscopic methods, namely Fourier transform infrared (FTIR) and Raman spectroscopies (RS), combined with the microscope can be applied in various field of medicine, including biomedical materials characterizations. The structural variations occurring due to the cancer development in cellular biomarkers, such as RNA, DNA, proteins, lipids, phosphates and carbohydrates, can be successfully detected in the collected spectra [4].

In these studies we discuss the data obtained for the two types of tumor tissues (inverted papilloma and salivary glands) using the afore mentioned methods. Fig. 1 shows the FTIR spectra of the salivary glands tissue recorded from two different areas. Additionally, the Amide I and lipids distribution maps are presented. The performed analysis allowed for identification of structural changes observed within the same cancerous tissues. Moreover, the spectral patterns due to the lipid regions suggest that there is a significant differentiation occurring in the various types of cancerous tissues. Acknowledgement: This research was performed using equipment purchased in the frame of the project co-funded by the Małopolska Regional Operational Program Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project No. MRPO.05.01.00-12-0/1513. References [1] I. Schwentner, P. Obrist, W. Thumfart et al., Acta Oto-Laryngol. 126, 340-345 (2006). [2] N. Toshitaka, S. Eiichi, I. Rie et al., ActaHistochem. Cytoc. 45, 269-282 (2012). [3] N. Sadeghi, S. Al-Dhahri, J. J. Manoukian, The Laryngoscope 113, 749-53 (2003). [4] K. Gajjar, L. D. Heppenstall, W. Pang et al., Anal. Methods 5, 89–102 (2013).

Figure 1. FTIR spectra of salivary glands from two different areas (A, B) together with the lipid (1740 cm-1; C) and Amide I (1650 cm-1; D) intensity maps.

1742

1657

1551

1457

1236

1084

1800 1600 1400 1200 1000

Abs

orba

nce

Wavenumber [cm-1]

A

B

C

D

AFM

L

1 Depar

2Dep

% Curre

Atomic Finfrared allows iresolutioand imagshow thspecific Figure 1absorptioassignedobservatFigure 1The mapranging the peakand othe1C and 1A-E. Texpectedindividu

Acknowthe proje2014/15/research managedL.Q.). Referenc[1] A. Da

M-IR Spec

Luca Qua

rtment of Ph

partment of P

ent Address

Force Microspectral reg

in principle on. We now uging of subc

hat the technsubcellular sA shows anon peak of

d to polymetion of cytoskB shows a mp reveals a in size from

k absorption er micrometr1D). Figure

The spectrumd for proteoliual organelles

wledgement: Pect co-funded/B/ST4/0483 and innovatd by the Nati

ces azzi and C. B P

ctromicrosand Cy

roni1, 2, *, K

hysical CheUn

ExperimentPolish Acads: Institute f

S

oscopy – Infrgion with a s

to performuse fixed fibellular structnique allowsstructures.

n AFM-IR mproteins. Th

erized actin keletal struct

map recordedrange of m

m about 100 nof phosphol

ric structures1F shows a

m displays ciposome-typs, cellular bio

Part of this red by the Mał39. Luca Quation programional Science

Prater, Chem.

scopy andytoskeletal

KatarzynaWojcie

mistry and niversity, PLtal Physics demy of Scifor MedicineSmith Walk,

*Luca.q

rared (AFM-spatial resolu

m studies of broblast cellstures, includis imaging w

map recorded he resultingfilaments o

tures by AFMd with 2920 c

micelles and nm to more lipid ester cas that may besample AFMcontributione particles. Wochemistry a

esearch has błopolska Regaroni has rec

m under the Me Center Pola

. Rev. 10.1021

d Imagingl Structur

a Pogoda2,

ech M. Kw

ElectrochemL-30060, Kof Complexiences, PL-3e and Engin, Philadelphquaroni@uj

IR) spectrosution better eukaryotic

s to demonstring vesicles, with intrinsic

with 1650 cg contrast ref the cytosk

M-IR inside acm-1 excitatiolipid dropletthan 1000 n

arbonyls, proe organelles

M-IR spectrus from both

We discuss thand the chem

been perform

gional Operateived fundin

Marie Skłodoand under PO

1/acs.chemrev

g of Vesiclres in Fibr

, %, Joannawiatek2

mistry, FacuKraków, Polax Systems, I31342, Krakneering, Unihia, PA, USj.edu.pl

copy allows than 50 nmcells with

rate the use omicelles, or

c contrast a

cm-1 excitatioeveals a fibekeleton. To a cell. on, at the absts distribute

nm. Excitatioovides additior fragment

um of one of h phospholiphese observa

mistry of the f

med using eqtional Prograng from the Ewska-Curie

OLONEZ co

v.6b00448 (20

Figure 1. Amapping of recorded wito the distribMap with 2distributionwith 1740 cvesicles andtopography AFM-IR spA, B, E - 5

les, Micellroblasts

a Wiltowsk

ulty of Chemand Institute of Nków, Polandiversity of PA

spectroscopm. [1] The hi

subcellular of AFM-IR

rganelles andand chemica

on, corresponer structure our knowle

sorption peakd throughou

on at 1740 cmonal images

ts of stackedf the particlespid and proations in termfixation proc

quipment puram Measure European Ungrant agreem

ontract 2016/

016)

AFM-IR spectrf fixed Detroitith 1650 cm-1 bution of prot920 cm-1 exci

n of long chaincm-1 excitationd organelle-likof the sample

pectrum of a siµm; C, D - 1.

Poster S

les, Organ

ka-Zuber2

mistry, Jagi

Nuclear Phyd Pennsylvani

pic studies inigh spatial rand single-in spectromi

d the cytoskeal characteriz

nding to thewhich is te

edge this is

k of long acyut the cytoplm-1, correspo of vesicles,

d membraness observed in

otein componms of the propcess.

rchased in fra5.1. Project N

nion’s Horizoment No. 665/21/P/ST4/01

romicroscopyt 551 fibroblasexcitation, co

teins in the samitation, showinn lipids. C andn, showing detke structures. Ee area in A andingle particle. 5 µm.

Session

nelles

2,

iellonian

ysics,

ia, 3340

n the mid-resolution organelle icroscopy eleton.We zation of

Amide I entatively

the first

yl chains. lasm and onding to micelles

s (Figures n Figures nents, as perties of

ame of No.

on 2020 5778, 1321 (to

y and sts. A: Map orresponding mple. B: ng the d D: Maps tails of E: d B. F: Scale bars:

N

AdriMar

We aimcontribuphosphodemonsmembra[1]. Werequiredspectralheadgrophosphaphosphoand biointernalmappingtopograand muFigure describe

Acknowresearch managedL.Q.)

Referen[1] I. Am[2] Z. Ars

Nanoscale MPhosp

ian Cerneriusz Kęp

1Ne2AGH Un

3Jagie

m to use nanutes to the polipid bilastrated the anes using te now showdto measurel range. Weoup in thisate stretchinolipids and omolecules probes tog of the p

aphy.We shoultiple phosp1C). This e the bioche

wledgement: T and innovatd by the Nati

nces menabar et al. N

sov, L. Quaro

Mid-infrapholipid B

scu1, Michczyński3, M

Mari

aspec Gmbniversity of ellonian Un

noscale IR sproperties oyers as mpossibility

the sSNOMw, for the fe spectra of observe th

s spectral rng modes (Ftheir surrouexternal to study the

phosphate aow that wepholipid biwork open

emistry of c

This project tion programional Science

Nature Commoni, Biochim. B

ared SpectBilayers as

hał SzuwaMagdalenia Nowak

H, BunsensScience and

niversity, Fa

pectroscopyof cell memmodels of

to recordM (scattering

first time, tphospholipe main absoregion aboFigure 1A). unding envio the bilaye

biochemicabsorption be can achievilayers and ns the way cell membra

has receivedm under the M

e Center Pola

m4, 1-9 (2013)Biophys. Acta

troscopic s Models o

arzyński2, na Wytrwaowska3, L

strasse 5, 82d Technolo

aculty of Ch

y to understmbranes. In

biological nano-FTIR

g-mode Nearthat the sampids in indivorption ban

ove noise lThese band

ironment, iner [2]. Thecal propertiband at 10ve sufficienother structo future s

anes and mo

d funding froMarie Skłodoand under PO

. a -Biomembra

Figumappatc(redthe imaat topodiscbila

and Imagof Biologi

Urszula Kał-Sarna2,

Luca Quaro

2152 Martingy, ACMiN

hemistry, PL

tand how ththis prelim

membranR spectra or Field Optime configurvidual bilayends of the dilevel, includs are sensincluding theerefore, theyies of the 070 cm-1 sint contrast tctures, suchstudies thatodel systems

m the Europwska-Curie

OLONEZ co

anes 1778, 880

ure 1. Nano Fps of supportches. A. Nanod) and two do

phosphate grage of a region1070 cm-1 reography. Thecriminate betayer patches. T

ging Studiical Memb

Kwolek3, PSzczepanoni3*.

nsried, GermN, PL-30059L-30387, Kr

he moleculaminary worknes. Past wof the proteical Microscration provers in the 80ipalmitoylphuding the eitive to the e aqueous my are amonmembrane.imultaneouto discriminh as liposomt use nano-s.

pean Union’sgrant agreem

ontract 2016/

0-889 (2008).

FTIR spectra ated DPPC sino FTIR spectrouble bilayersroup region. n of the sampecorded simu difference intween singleThe circle indi

Poster S

ies of Singbranes

Paweł Wyn Zapotocz

many 9, Kraków raków

ar structure k, we use suwork has ein complecopy) confi

vides the se00 cm-1 – 20hosphatidylester carbointeraction

medium, othng the mos. We also sly with thnate betweemes(Figure IR spectros

Horizon 202ment No. 665/21/P/ST4/01

and single wavngle and mulra of a singles (blue and gB. AFM tople. C. Nano IRultaneously wn intensity al, double anicates a liposo

Session

gle

dro3, zny3,

of lipids upported

already ment of

iguration ensitivity 000 cm-1 l choline

onyl and between

her lipids st useful perform

he AFM en single

1B and scopy to

20 5778, 1321 (to

velength ltibilayer e bilayer green) in pography R image with the llows to

nd triple ome.

Poster Session

“Checker-FTIR”: Applying FTIR to Checker Board Assays for Drug Mixtures Inhibition Tests

Luca Roscini1, Laura Corte1, Debora Casagrande Pierantoni1, Daniele Fioretto2,3,

Vincent Robert4, Giuseppe Bellisola5 and Gianluigi Cardinali1,2,

1Department of Pharmaceutical Sciences, University of Perugia – Italy

2CEMIN Excellence Research Center – University of Perugia, 3Department of Physics and Geology- University of Perugia – Italy,

4Westerdijk Institute of Fugal diversity – Utrecht –NL, Biomedical Research Center – 5associated to INFN - LNF, SINBAD Lab – Italy.

The idea of using FTIR as a tool to obtain fingerprints of microorganisms was introduced more the twenty years ago [1]. This technique has been soon proposed as a metabolomic fingerprinting tool [2] able to portray the metabolic status of microbial cells. Several studies have also demonstrated that the FTIR technique can be used to detect a stressing situation or an illness with a good sensitivity [3, 4]. The classical microbial assays for stress detection are based on the ability of cells to grow in the specific tested conditions, giving a clear and easy-to-understand binary result: growth or no growth. This is the case of MIC microdilution test, where microbial cells are exposed to a certain number of different concentrations of specific drugs to test their inhibitory ability [5]. These types of bioassay represents an important tool to determine the biological effect caused by chemical, physical or biological agents and are even more important in environmental sciences in which complex mixtures of toxic compounds are present [6]. In presence of binary mixtures of drugs, the checker board method is used that consists in combining serial dilutions of the two compounds in the rows and in the lines of a microtiter plate. Synergistic or antagonistic effects are therefore visualized according to the pattern of inhibition and no inhibition wells. Among the methods used to detect in vitro synergy/antagonism between different compounds, the checkerboard microdilution method is one of the most widely used in current medical and environmental microbiology research [7]. The highlighting of a metabolic change is the basis of every bioassay, but not many tests are designed to quantify and classify the changes detected, though these pieces of information are useful to evaluate not only the presence of toxic agents but also which type of action/interaction they exert [4]. In this study, we propose the application of FTIR spectroscopy to a checkerboard microdilution procedure to display the metabolic changes occurred after the exposition to a mixture of compounds and to highlight additive, synergic or antagonistic behaviors. References [1] D. Naumann, D. Helm and H. Labischinski, Nature 351; 81-82 (1991). [2] O. Fiehn, Comparative and Functional Genomics 2; 155-168 (2001). [3] L. Corte, P. Rellini, L. Roscini, F. Fatichenti and G. Cardinali, AnalyticaChimicaActa 659; 258-265 (2010). [4] L. Corte, L. Roscini, C. Zadra….and G. Cardinali, Food Chemistry 134, 1327-1336 (2012). [5] M. Cuenca-Estrella, W. Lee-Yang, M. A. Ciblak… and J. L. Rodriguez-Tudela, Antimicrobial Agents and

Chemotherapy 46, 3644-3647 (2002). [6] A. Stewart and J. Carter, Environmental Geochemistry and Health 31, 239-251 (2009). [7] R. L. White, D. S. Burgess, M. Manduro and J. A. Bosso, Antimicrobial Agents and Chemotherapy 40, 1914-

1918 (1996).

Poster Session

A Multimodel Approach to Babesia bovis Diagnosis

Anja Rüther1, William Poole2, David Perez Guaita1, Philip Heraud1,2,3, Brian Cooke2,3, Bayden Wood1

1Centre for Biospectroscopy, Monash University, 3800, Victoria, Australia

2Department of Microbiology, Monash University, 3800, Victoria, Australia 3Biomedical Discovery Institute, Monash University, 3800, Victoria, Australia

Babesia, an apicomplex parasite similar to the malaria parasite Plasmodium, is the second most common bloodstream parasite found in mammals [1]. It is transmitted to its vertebrate host by tick bites or blood transfusions. Babesia bovis infects cattle herds in tropical regions worldwide and leads to mortalities, abortions and a reduction in meat and milk production. Tick control, prevention and treatment are expensive and there are only cost-intensive (polymerase chain reaction) or human error-prone (microscopy) diagnosis methods available [2]. A sensitive and reliable method for the diagnosis of babesiosis that enables the detection of low parasitemia is highly desired.

A number of optical spectroscopic techniques to detect the human malaria parasite were explored in our lab, including Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy as a non-subjective diagnosic tool [3].When examining the case of human malaria infection, a metabolite of the Plasmodium, namely hemozoin, simplifies the detection of Plasmodium as it shows a characteristic infrared signature.

Here, we demonstrate the applicability of spectroscopic methods for the detection of anon-hemozoin producing parasite: B. bovis.

1) First, we established ATR-FTIR spectroscopy combined with chemometric data analysis for the detection of B. bovis in cultured cattle red blood cells (cRBCs). This method can be transferred to the field as a simple and quick diagnostic tool for babesioses. As ATR-FTIR on blood samples is usually challenged by the background absorbance of blood components, blood samples were lysed thereby removing blood components. This increased the detection sensitivity from 77.3% to 92.0% for 0.25% parasitemia. We detected samples with down to 0.001% parasitemia with a sensitivity and specificity of 74.2% and 80.6%, respectively. 2) Using atomic force microscopy (AFM) IR, which allows for the collection of IR spectra with a nanoscale spatial resolution, we could assign spectral features that are characteristic for aparasite measured directly inside of cRBCs, i.e. DNA and lipid IR-bands, for example. 3) Synchrotron-based Focal Plane Array (FPA) IR allows for IR imaging with the high intensity of synchrotron radiation. Averaging over the pixels in an image of B. bovis infected cRBCs and uninfected cRBCs yields spectra corresponding to a whole infected or uninfected cRBC, respectively.

The spectral changes fromATR-FTIR that account for the presence of B. bovis in cRBCs were confirmed by the use of AFM-IR directly on the parasite inside of a red blood cell and by synchrotron-based FPA-IR on B. bovis infected and uninfected cRBCs. We show here the proof of principle of using IR spectroscopic techniques for diagnosing the threatening disease babesiosis. References [1] L. Schnittger, A. E. Rodriguez, M. Florin-Christensen, D. A. Morrison, Infec Genet and Evol. 12, 1788–1809

(2012). [2] R. Bock, L. Jackson, A. De Vos, W. Jorgenson, Parasitology 129, S247–S269 (2004). [3] A. Khoshmanesh, M. W. Dixon, S. Kenny, L. Tilley, D. McNaughton, B. R. Wood, Anal Chem 86, 4379

(2014).

Poster Session

High-throughput Raman Spectroscopy of Single Cells

Iwan W. Schie1, Saif Abdullah Mondol1, Jan Rüger1, Christoph Krafft1, and Jürgen Popp1,2

1Leibniz Institute of Photonic Technology (IPHT), Albert-Einstein-Straße 9, Jena, Germany

2Institute of Physical Chemistry, Friedrich Schiller University Jena, Helmholtzweg 4, 07743 Jena, Germany

In recent years there have been a significant number of novel and promising biomedical and clinical applications using Raman spectroscopy [1]. Specifically label-free single cell analysis has shown interesting opportunities for biomedical research [2,3]. It was shown that not only can eukaryotic cells can be identified label-free, but also the interaction of drugs on cells can be investigated. While many publications have shown very intriguing results, the implementations suffer from distinct problems, which hamper a broader implementation of this method. Due to the complex data acquisition procedure the experiments are usually performed on a very small number of cells, typically on the order of hundred cells, and rarely on mixed populations [4]. This, however, results questionable statistical results and makes the experiments difficult to translate to real world scenarios. We have addressed this problem by implementing a fully automated data acquisition approach, which allows the measurement of thousand of cells in a short time. This implementation results in many new and exciting applications. The new approach was applied to perform a label-free white blood cell differentiation and showed comparable results to standard machine counting methods; with Raman-measurements performed on more than 20,000 cells. Furthermore, the setup was successfully tested for a rapid time series of drug cells interaction measurements, and the identification of tumor cells in a mixture with background cells. These results will be presented.

Acknowledgments: This work is supported by the EU-funded project MIB (No 667933) the European Union within the FP7 collaborative project CanDo (610472) and by the Leibniz Association through the project Hyperam (SAW-2016-IPHT-2). References [1] C. Krafft, I. W. Schie, T. Meyer, J. Popp, Chem. Soc. Rev. (2015), DOI 10.1039/c5cs00564g. [2] I. W. Schie, L. Alber, A. L. Gryshuk, J. W. Chan, Analyst 139, 2726–2733 (2014). [3] I. W. Schie, R. Kiselev, C. Krafft, J. Popp, Analyst 141, 6387–6395 (2016). [4] C. Beleites, U. Neugebauer, T. Bocklitz, C. Krafft, J. Popp, Anal. Chim. Acta 760, 25–33 (2013).

Poster Session

FTIR Spectroscopy for HT-screening and Monitoring of Single Cell Oil Production

Julie Eymard1, G. Kosa1, B. Zimmermann1, V. Tafintseva,

S. Dzurendova1, A. Kohler1, V. Shapaval1

1 The Faculty of Science and Technology, NMBU, Ås, Norway

Oleaginous microorganisms are accumulating up to 80% of their biomass in the form of lipids and are considered as alternative source of lipids for various applications such as food supplements, animal feed and biodiesel. The selection of suitable microbial lipid producers and substrates and the development of cultivation conditions are among the main challenges for cost-effective production of microbial-based single cell oils. In order to meet these challenges, massive, rapid and precise screening of total lipid content and fatty acid profile is needed. In on-going and previous research projects in Norway, we have been developing and using FTIR spectroscopy extensively for screening of microbial cell products to develop processes for the production of single cell oils and other high-value products [1], [2]. In the current study we present how FTIR spectroscopy can be applied for screening of oleaginous microorganisms and bioprocess optimization for single cell oil production in oleaginous fungi. A huge amount of oleaginous filamentous fungi and yeasts were cultivated on various substrates (glucose, glycerol, animal fat) and under different cultivation conditions (time and temperature). FTIR spectroscopy was applied for the prediction of the total lipid content and profile (saturated, monounsaturated and polyunsaturated fatty acids) in the fungal biomass as well as for monitoring substrate consumption (glucose and fat) and metabolite release (citric acid) [3]. For predicting the fatty acid composition of microbial cells we calibrate partial least squares regression (PLSR) models for a set of strains where fatty acid composition was measured by GC reference analysis. References [1] NFR project: “Single cell oil PUFA production by food rest materials – SingleCellOil” No 234258. NFR

project: Bioconversion of low-cost fat materials into high-value PUFA-Carotenoid-rich biomass, No: Projectnr: 268305. NFR project: Bio4Fuels Norwegian Centre for Sustainable Bio-based Fuels and Energy, Projectnr: 257622 financed by the Research Council of Norway.

[2] V. Shapaval, N. K. Afseth, G. Vogt, A. Kohler A, “Fourier Transform Infrared Spectroscopy for the prediction of fatty acid profiles in Mucor fungi in media with different carbon sources”, Microbial Cell Factories, (4), 13, 86 (2014).

[3] G. Kosa, A. Kohler, V. Tafintseva, B. Zimmerman, K. Forfang, D. Tzimorotas, N. K. Afseth, K. S. Vuoristo, S. J. Horn, J. Mounier, V. Shapaval, “Duetz Microtiter plate system combined with FTIR spectroscopy for the screening of oleaginous fungi and high-throughput monitoring of lipogenesis”, Microbial Cell Factories (16), 101 (2017), DOI: 10.1186/s12934-017-0716-7.

    Poster Session

IR Nanopolarimetry: Anisotropy in Biomolecular Assemblies and Thin Biofilms

T. Shaykhutdinov, A. Furchner, and K. Hinrichs

Interface Analytics Research Department, Leibniz-Institut für Analytische Wissenschaften –

ISAS – e.V., Schwarzschildstr. 8, 12489 Berlin, Germany Direct identification of biomolecular anisotropy at the nanoscale is of fundamental importance for understanding peptide self-assembly mechanisms, controlling protein misfolding and aggregation, synthesis of biomimetic materials, and biofunctionalization of interfaces. We present IR nanopolarimetry [1], a label-free and non-invasive spectroscopic method that provides insights into anisotropic structural organization by correlating morphology with internal chemical composition, intermolecular interactions, and molecular ordering with ≤ 30 nm spatial resolution.

This high-sensitivity approach to nanoscale characterization of biomolecular aggregates and thin biofilms is based on AFM-IR and measures anisotropic vibrational signatures of the molecules in seconds. The commercially available set-up (Anasys Instruments nanoIR2-FS) uses an AFM tip to directly probe IR absorption via thermal expansion of the sample under p- and s-polarized low-power QCL pulses. In contrast to other polarization-dependent nanospectroscopic techniques, the obtained IR nanopolarimetric information is not limited to the surface of the sample, and provides both the in-plane and the out-of-plane optical properties with straightforward data interpretation.

This presentation will highlight the broad applicability of IR nanopolarimetry to biomolecular diagnostics by focusing on the following phenomena: oriented Concanavalin A aggregation upon adsorption, polarization-dependent biosensing of a peptide nucleic acid [2] on biofunctionalized graphene films [3], and anisotropic growth mechanisms of supramolecular porphyrin aggregates [1]. The analysis of the nanoscale anisotropy is supported by vibrational (DFT) and electrodynamic (FDTD) calculations. References [1] T. Shaykhutdinov, S. D. Pop, A. Furchner, K. Hinrichs, ACS Macro Lett. 6, 598–602 (2017). [2] F. Rösicke, M. A. Gluba, T. Shaykhutdinov, G. Sun, C. Kratz, J. Rappich, K. Hinrichs, N. H. Nickel, Chem.

Commun., DOI: 10.1039/C7CC03951D (2017). [3] K. Hinrichs, T. Shaykhutdinov, C. Kratz, F. Rösicke, C. Schöniger, C. Arenz, N. H. Nickel, J. Rappich, in: K.

Wandelt (ed.): "Encyclopedia of Interfacial Chemistry: Surface Science and Electrochemistry" Elsevier (accepted, 2017).

Poster Session

Single Factor Stress Response Studies of Mcf-7 Breast Cancer Cells by FTIR Spectroscopy

Karlis Shvirksts1, Mara Grube1, Dominyka Dapkute2, Marija Matulionyte2,

Ricardas Rotomskis2, Elina Zandberga3, Aija Line3

1Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia 2Laboratory of Biomedical Physics, National Cancer Institute, Vilnius, Lithuania

3Latvian Biomedical Research and Study Centre, University of Latvia, Riga, Latvia The response of cells to various growth factors via the biochemical composition is well

known and has been studied by FT-IR spectroscopy. Lately there has been an increase of studies of nanoparticlesas promising agents for targeted therapy. It also has been shown that cell cultivation under hypoxic conditions enhancesthe uptake of nanoparticles. Despite propitious prospects, the stress response of cells cultivatedin presence of nanoparticles or under hypoxic conditions remains poorly understood.

In this study breast cancer cells MCF-7 were used to evaluatethe single factor stress response. Three stress factors were used:pure BSA, BSA-encapsulated photoluminescent gold nanoclusters (Au-BSA NCs) [1], and hypoxic conditions. FT-IR spectra of ~200’000 cells were acquired by HTS-XT microplate reader (Bruker optics, Ettlingen, Germany). Quantitative analysis of macromolecular composition was carried out as in [2].

MCF-7 cells showed little response to the presence of BSA or Au-BSA NCs. Compared with control, the content of carbohydrates in cells incubated with BSA or Au-BSA NCs was slightly lower - 10.12% and 9.52% of dry weight (dw) compared to 12.7% in control. The content of nucleic acids and proteins was slightly increased – from 7.92% and 63.42% to 9.02% and 64.64% dw, respectively. Lipid content increased from 5.96% to 6.21% dw for BSA, and 6.83% dw for Au-BSA NCs incubated cells. Hypoxic conditions induced stronger stress response. The content of carbohydrates, nucleic acids and proteins all slightly decreased to 12.14%, 7.58% and 61.07% dw, respectively. Whereas the lipid content increased 1.54 times from 5.96 to 9.21% dw.

Results showed minor difference in themacromolecular composition of cells incubated with BSA or Au-BSA NCs thus suggesting that Au-BSA NCs have no significant stress effect on cells and are relatively safe to use. However enhancing of nanoparticle uptake by hypoxic conditions needs further studies due to the remarkable effect on the total lipid content in cells.

Acknowledgment: This study was supported by the LCS joint project “Cancer-derived exosomes – a source of novel biomarkers and therapeutic targets for gastrointestinal cancers”.

References [1] V. Poderys et al., Lith. J. Phys. 56, 55–65 (2016). [2] M. Grube et al., Vibr. Spectr. 28, 277–85 (2002).

    Poster Session  

Fast Resonant Mie-scatter Correction Algorithm: Parameter Choice and Validation

J. Solheim1, T. Konevskikh1,2, A. Kohler1

1Department of Sciences and Technology (IMT), Norwegian University of Life Sciences,

1430 Ås, Norway. 2MWT Analytics, 1430 Ås, Norway

Infrared spectroscopy of micrometer-sized and approximately spherical structures, such as single cells, are affected by Mie scattering. During recent years, several methods have been proposed for retrieving pure absorbance spectra from such measurements.

In 2008 a method based on extended multiplicative signal correction (EMSC) was proposed, where an approximation formula for the Mie extinction was implemented in an EMSC model by a meta-model using principal component analysis (PCA) [1]. The approach was further developed to handle the so-called resonant case, where the real part of the refractive index undergoes fluctuations due to absorption [2]. This model is called resonant Mie Scatter EMSC (RMieS-EMSC). Recently, Konevskikh et. al. suggested a further improvement of the Bassan algorithm, as it could be shown that the corrected spectra are not strongly affected by the reference spectra used: the algorithm shows much clearer chemical features of the measured spectrum [3].

In this work, the algorithm by Konevskikh et. al. was further refined. We present a program for correcting absorbance spectra, where the RMieS-EMSC algorithm is implemented in MATLAB. In order to test the stability of the code, a set of apparent absorbance spectra was simulated. The pure absorbance spectra were based on the matrigel spectrum, and chemical information was altered by changing absorbance peak heights. Mie scatter contributions were estimated from experimentally obtained measurements [1]. Apparent absorbance spectra with similar Mie scattering features as the experimental data were simulated using the simulated pure absorbance spectra as input for the imaginary part of the refractive index. The obtained simulated apparent absorbance spectra have the following advantages: (1) the underlying pure absorbance spectra are known and corrected spectra can be directly compared to the pure absorbance spectra used for simulation, (2) the scattering features of the simulate apparent absorbance spectra resemble scattering features observed in experimentally obtained spectra. The simulated spectra were used to test the stability of the RMieS-EMSC algorithm and to refine the algorithm further. Results demonstrate high stability of the algorithm for a variety of parameter settings. Sensitivity towards the number of principal components used in the correction is reviewed. To some extent this number is affecting the correction in the amide region, but the impact is shown to be generally low. As the algorithm is an iterative process, both the stop criterion and measures to decrease computational time are reviewed. The algorithm’s ability to retrieve the true peak position of the amide I band is discussed. References [1] A. Kohler, J. Sulé-Suso, G. Sockalingum, M. Tobin, F. Bahrami, Y. Yang, J. Pijanka, P. Dumas, M. Cotte,

D. G. van Pittius, G. Parkes and M. Høy, Appl. Spectrosc. 62, 259-266 (2008). [2] P. Bassan, A. Kohler, H. Martens, J. Lee, H. Byrne, P. Dumas, E. Gazi, M. Brown, N. Clarke and P. Gardner,

Analyst 135, 268-277 (2010). [3] T. Konevskikh, R. Lukacs, R. Blümel, A. Ponossov and A. Kohler, FaradayDiscuss. 187, 235-257 (2016).

    Poster Session

ATR-FTIR Microplate Reader and Micromachined ATR Silicon Crystals

Lorenz Sykora, Anja Müller

Walter Schottky Institute, Technical University Munich, Am Coulombwall 4, Munich, Germany

The analysis of blood samples by Fourier Transform Infrared (FTIR) Spectroscopy to detect diseases like cancer1,2 or malaria3are an upcoming topic in the last decades. There are ongoing efforts to transfer infrared spectroscopy from research into clinics. Therefore, large databases for machine learning and calibration algorithms are needed. An automated ATR high-throughput device was developed to enable larger studies.

Left:Silicon ATR crystal with v-grooves on the bottom side. Middle: Bottom side of the microplate. Right: Prototype of ATR high-throughput device.

Cost-effective silicon ATR crystals introduced by Schumacher et. al4 where optimized for the integration into microplates and higher signal intensity. The fabrication makes use of proven processes of the semiconductor industry and leads to crystals which are up 50–100 times cheaper compared to conventional ATR crystals. The very short light path of just 1 mm gives access to the full spectral range of standard FTIR spectrometers, which also covers the fingerprint region. Spectra recorded with these silicon crystals are comparable to those of conventional single reflection elements. References [1] A. L. Mitchell, K. B. Gajjar, G. Theophilou, F. L. Martin, & P. L. Martin-Hirsch, “Vibrational spectroscopy

of biofluids for disease screening or diagnosis: translation from the laboratory to a clinical setting”, Journal of biophotonics 7(3-4), 153-165 (2014).

[2] J. R. Hands, K. M. Dorling, P. Abel, K. M. Ashton, A. Brodbelt, C. Davis, ... & M. J. Baker, J. Biophotonics 7, 189–199 (2014).

[3] A. Khoshmanesh, M. W. Dixon, S. Kenny, L. Tilley, D. McNaughton & B. R. Wood, “Detection and quantification of early-stage malaria parasites in laboratory infected erythrocytes by attenuated total reflectance infrared spectroscopy and multivariate analysis”, Analytical chemistry 86(9), 4379-4386 (2014).

[4] H. Schumacher, U. Künzelmann, B. Vasilev, K. J. Eichhorn & J. W. Bartha, “Applications of Microstructured Silicon Wafers as Internal Reflection Elements in Attenuated Total Reflection Fourier Transform Infrared Spectroscopy”, Applied spectroscopy 64(9), 1022-1027 (2010).

Poster session Investigating the uptake and response of hMSC cells exposed to Falcarindiol

Anders R. Walther1, Richard H. Cowie2, Jes Linnet2, Dennis Høj2,

Jakob D. Jepsen2, Rime B. El-Houri1, Eva A. Christensen1, Morten Ø. Andersen1, Martin A. B. Hedegaard1

1Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

2The Maersk Mc-Kinney Moller Institute, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

Falcarindiol is a dietary polyacetylene that is found in many food plants of the Apiaceae family and is typically isolated from carrots. Falcarindiol have shown anti-cancer effects e.g. reducing formation of neoplastic lesions in rats1. Falcarindiol have also been shown to have a positive effect on diabetes where falcarindiol increased peroxisome proliferator-activated receptor (PPAR)γ-mediated transactivation significantly2. The biological mechanisms on a single cell level is however unknown.

Experimental: To investigate the uptake mechanism of falcarindiol we have designed a Raman imaging experiment of hMSC cells at different time points of exposure to 10 µm falcarindiol in the cell culture medium. Cells were cultured on glass and Exposed (FC) and control cells were fixed at 0h, 1h, 5h and 24h in three biological replicates.

Raman imaging were performed using a in house build Raman imaging setup using a 532nm Laser. For imaging a 100x NA=1 water immersion objective was applied resulting in an effective spatial resolution around 800nm. Images were collected with 60mW at the sample and integration times of 1s per spectrum. For each control and FC a minimum of three cells were imaged for each time point and biological replicate.

Data Analysis: As cells were measured on glass, the glass background was a major issue. This was corrected using EMSC-SIS with references extracted from each individual dataset. The images where then analysed using the N-FINDR spectral unmixing algorithm using three to five endmembers per image. For each image, a combined false color image was constructed based on abundance values. The method is described in further detail by Hedegaard et al3.

Results: For the time points 0h and 1h there were in general no change in cell composition. The same was the case for the 24h control cells which confirms that no greater changes are occurring during the 24h for our control group. After 5 and 24h exposure to falcarindiol we observe significant changes by the formation of cholesteryl linoleate (CLA) droplets in the cytoplasm. In most 5h we see this trend and after 24h all cells show CLA.

Conclusions: We can conclude that after 24h falcarindiol have a significant impact on hMSC cells, resulting in a major increase CLA production. This indicates potential impact on sterol pathways and possible indirect mechanisms for the effects shown on cancer and diabetes.

[1.] Kobaek-Larsen, M., El-Houri, R. B., Christensen, L. P., Al-Najami, I., Fretté X., Baatrup G., (2017) Food

& Function. DOI: 10.1039/c7fo00110j.

[2.] El-Houri,R. B., Kotowska, D., Christensen, K. B., Bhattachary, S., Oksbjerg, N., Wolber, G., Kristiansen K., Christensen, L.P. (2015), Food & Function , 54, (6), 2135–2144.

[3.] Hedegaard, M. A. B., Bergholt, M. S., Stevens, M. M., (2016), J. Biophotonics (9), No. 5, 542–550

Poster Session

Raman Microspectroscopy for Non-invasive, Three-dimensional Analysis of Biofilms

R. Weiss, R. Niessner, M. Elsner, M. Seidel, N. P. Ivleva

Institute of Hydrochemistry and Chair of Analytical Chemistry and Water Chemistry,

Technical University of Munich, Marchioninistr. 17, 81377 Munich, Germany Confocal Raman Microspectroscopy (RM) enables the three-dimensional, chemical characterization of transparent samples with particles in the µm range. Especially sensitive and complex structures, e. g. biofilms, are suited for the analysis by RM. Few sample preparations are needed: mechanical strain does not significantly affect the sample during the measurement and water does not interfere with the spectroscopic analysis. In their natural habitat, microbial communities tend to develop biofilms. Aquatic microorganisms predominantly occur in this form of appearance [1]. Non-invasive, three-dimensional, potential time-resolved analyses of biofilms offer the potential for access to fundamental information about interactions between microorganisms, the occurrence of pathogens or the flow of water quality-related pollutants.

Our work with artificial biofilm models indicates the high spatial resolution of RM in all three dimensions. Microparticles and bacterial cells (e. g. E. coli) could be chemically differentiated 0.5 mm below the sample surface. Further, the combination of Stable Isotope Probing (SIP) and Surface-Enhanced Raman Spectroscopy (SERS) was explored as shown in Figure 1. The ability to detect the incorporation of stable isotopes features another striking advantage of the RM for the analysis of biological samples [2-4]. By evaluating red-shifted signals three-dimensionally it is possible to gain a more detailed insight into the fate of stable isotope labeled compounds or relations between microbial communities. Special focus is supposed to be on Legionella pneumophila-containing biofilms, which is the causative germ of the Legionnaires’ disease. Unicellular host organisms not only facilitate growth, survival and recovery of Legionella but also enhance virulence of this pathogen [5]. The three-dimensional analysis potential of RM might promote new leads into this parasitic relationship and its impact. References [1] J. W. Costerton, K. J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, T. J. Marrie, Ann Rev

Microbiol. 41, 435-464 (1987). [2] Y. Wang, W. E. Huang, L. Cui, M. Wagner, Curr Opin Biotechnol. 41, 34-42 (2016). [3] P. Kubryk, J. S. Kölschbach, S. Marozava, T. Lueders, R. U. Meckenstock, R. Niessner, N. P. Ivleva, Anal

Chem. 87, 6622-6630 (2015). [4] M. Chisanga, H. Muhamadali, R. Kimber, R. Goodacre, Faraday Discuss. DOI: 10.1039/C7FD00150A

(2017). [5] U. Scheikel, H.-F. Tsao, M. Horn, A. Indra, J. Walochnik, Parasitol Res. 115, 3365-3374 (2016).

Figure 1. SERS map of an artificial biofilm (12C and 13C labeled E. coli with Ag nanoparticles in an agarose matrix) and embedded SERS spectra of single 12C and 13C E. coli cells.

Poster Session

Influence of CO2-Concentration on Raman Spectra of Bacteria

Christina Wichmann1,2, Petra Rösch2,3, Jürgen Popp1,2,3

1Institute of PhotonicTechnology Jena, Albert-Einstein-Straße 9, 07745 Jena, Germany 2InfectoGnostics Research Campus Jena, Philosophenweg 7, 07743 Jena, Germany

3Institute of Physical Chemistry, Friedrich-Schiller-University Jena, Helmholtzweg 4, 07743 Jena, Germany

The lung covers a large area up to 140 m2 [1]. Although this surface is exposed to the environment the lung seemed to be sterile for a long time, since their microorganisms could not be cultivated by standard cultivation methods.This theory could be disproved by cultivation independent methods such as PCR. Today the lung is known to harbor a variety of bacteria, viruses, fungi and phages [2-4]. Nevertheless, the knowledge of the lung microbiome shows big gasps. Raman microspectroscopic analyses of bronchoalveolar lavages can be a tool to get a better insight in the lung microbiome. To enable a Raman microspectroscopic characterization of the lung microbiome it is necessary to simulate the lung environment as best as possible. Therefore the CO2 concentration must be considered, since during breathing the content of CO2 changes constantly. Even general health of the lung influences the content of CO2. To simulate this situation, we incubate bacteria with different concentrations of CO2 to show the influence during growth on the Raman spectra. Acknowledgement: Financial support of the Leibniz project "The lung microbiota at the interface between airway epithelium and environment" (SAW-2016-FZB-2) is greatly acknowledged. References [1] P. Gehr, M. Bachofen, and E. R. Weibel, "Normal Human Lung - Ultrastructure and Morphometric

Estimation of Diffusion Capacity", Respiration Physiology 32(2), 121-140 (1978). [2] L. N. Segal et al., "Enrichment of the lung microbiome with oral taxa is associated with lung inflammation of

a Th17 phenotype", Nat Microbiol 1, 16031 (2016). [3] D. Willner et al., "Metagenomic Analysis of Respiratory Tract DNA Viral Communities in Cystic Fibrosis

and Non-Cystic Fibrosis Individuals", Plos One 4(10) (2009). [4] E. S. Charlson et al., "Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after

lung transplant", Am J Respir Crit Care Med 186(6), 536-45 (2012).

Poster Session

Fiber Optic Probe-Based Raman Imaging Using Positional Tracking

Wei Yang1, Iwan W. Schie1, Jürgen Popp1,2

1Leibniz Institute of Photonic Technology Jena, Albert-Einstein-Straße 9, 07745 Jena, Germany

2Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Helmholtzweg 4, 07743 Jena, Germany

Raman spectroscopy canprovide label-free spectroscopic fingerprint information, which makesit a highly valuable tool for biomedicaldiagnostics [1].For example, imaging of tissue samplescan providevisual information about the distribution of molecular components of the sample and can be used to detect, diagnose, and delineate tumor and normal tissues [2, 3]. Fiber-based Raman spectroscopy has been shown to provide superb identification ex vivo an in vivo of tumor grades. For in vivo applications it would be very advantageous to provide imaging information, this, however, is not straightforward using a fiber-probe [4].

Because most implementations of Raman spectroscopy provide only information from the focus spot, Raman images are created by mechanical sample or beam scanning approaches. This makes the scanning setup rigid and not easily movable, and restricts imaging applications directly at the patient. Here, we present a newly developed Raman-probe based imaging method, using traditional fiber-optical probes computational image processing [5]. Combining the simultaneous measurement of position information with spectroscopic Raman information, allows acquiring Raman imagesin a short time from large tissue samples. The proposed approached allows the access to any surface and the acquisition of Raman images from those surfaces. The chemical information canalsobe overlaid during the acquisition with the brightfield imaging information on a computer screen to create an augmented reality image of the biochemical distribution on a sample surface.This method allowsto easily distinguish borders of different biomolecular composition and can be extended to clinical applications of tumor border delineation and so improve tumor removal. References [1] M. Schmitt and J. Popp, J. Raman Spectrosc. 37, 20-28 (2006).

[2] C. Krafft, M. Schmitt, I. W. Schie, D. Cialla-May, C. Matthäus, T. Bocklity and J.Popp, Angewandte Chemie International Edition (2016).

[3] I. Latka, S. Dochow, C. Krafft, B. Dietzek and J. Popp, Laser &Photo. Rev. 7(5), 698-731 (2013). [4] C. Krafft, S. Dochow, I. Latka, B. Dietzek and J. Popp, biomedical spectrosc. and imaging 1, 39-55 (2012).

[5] RPB Laboratory Probe, InPhotonics, Inc., 111 Downey St., Norwood, MA 02062, USA

    Poster Session

Analysis of Plant Tissues Using Vibrational and Other Spectroscopic Methods and Multivariate Approaches

I. Zeise1, Z. Heiner1,2, M. Joester1,3, S. Diehn1,3, V. Rodriguez1,2,4,

F. Emmerling3, R. Elbaum4, and J. Kneipp1,2,3

1Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin

2School of Analytical Sciences Adlershof SALSA, Humboldt-Universität zu Berlin, Albert-Einstein-Straße 5-11, 12489 Berlin

3BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Straße 11, 12489 Berlin,

4The Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel

Understanding the structure and function of plant tissues requires the combination of analytical tools that govern different scales, ranging from the micro-morphology to the molecular composition, and that elucidate the interaction of organic and inorganic materials. While Raman spectroscopy as a vibrational method allows to investigate the structure and chemical composition of plant tissue sections, second harmonic generation (SHG) and fluorescence microscopy give complementary insight into the orientation, morphology, and heterogeneity of the distribution of the tissue building blocks at the microscopic level. Raman, Here, Raman microscopy was combined with SHG, two-photon excited fluorescence (2PF), [1] and scanning electron microscopy (SEM-EDX) to investigate several different plant tissues including e.g., pollen grains and cells walls in plant sections, and to image their composition and structure. Using multivariate techniques, specificallyhierarchical cluster analysis (HCA) andprincipal component analysis (PCA) on the Raman data sets, histological characterization of these tissues is possible. In pollen tissues, differentiation of histological substructures (pollen grain center, pollen tube shank or apex) is possible using PCA, and is an important prerequisite in order to identify small biological differences brought about by altered physiological situations [2]. Similarly, we present an approach to refold subspaces of PCA scores plots so that a visual identification of physiological differences is possible from multivariate tissue maps. As an example, we discuss the influence of silica both in the germination medium in pollen grains, and during the growth of whole plants. Silica also plays an important role in plants, as it can form microscopic silica deposits, called phytoliths. Variations in the silica nanostructure between the different phytolith cell types were observed using both, Raman techniques and SEM-EDX. We conclude thatonly the combination of all these different analytical tools can give a full picture of the influence of specific growth conditions on the structure of complex plant tissues. Acknowledgement: We thank D. Lajkó and V. Tarabykin (Charité Berlin) for providing access to the vibratome, S. Holz and C. Büttner (HU) for cucumber plants, and O. Markovich (The Hebrew University of Jerusalem) for sorghum seeds. We acknowledge S. Seifert for providing a MATLAB script for PC analyses, P. Lasch for CytoSpec software, and T. Schmid for help with SEM-EDX. Financial support from ERC Grant No. 259432 (Z.H., J.K.), DFG (GSC 1013 SALSA, Z.H.), Israel Science Foundation grant 534/14 (R.E.), and Einstein Stiftung Berlin (I.Z., M.J., F.E., R.E., J.K.) is gratefully acknowledged (grant A-2011-77). References [1] Z. Heiner; I. Zeise; R. Elbaum, J. Kneipp, submitted. [2] M. Joester; S. Seifert; F. Emmerling; J. Kneipp, J. Biophot. 10 (4), 542–552 (2016).

Poster Session

Comprehensive Vibrational Characterization of the Interaction of Liposomes and Gold Nanoparticles

Vesna Živanović, Freeda Yesudas, Zsuzsanna Heiner, Christoph Arenz,

Janina Kneipp

Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany

School of Analytical Sciences Adlershof (SALSA), Albert-Einstein-Str. 5-9, 12489 Berlin, Germany

Liposomes are important supramolecular structures with a wide range of applications in biophysics and medicine, e.g., as carriers for various therapeutic agents.In this context, the combination of d liposomes with gold nanoparticles can provide new composite systems, e.g., to create new drug delivery platforms [1]. Currently, many liposome-nanoparticles drug delivery systems are under investigation. In spite of the efforts, only a small number reaches the clinical investigation stage, one reason for this being the poorly investigated interactions between the components of the potential delivery system. Here, we aim at thecharacterization of potential liposomal carriers from different perspectives provided by complementary vibrational characterization. Surface enhanced Raman scattering can provide detailed spectroscopic informationabout the structure and interaction of liposomes with gold nanoparticles, while broadband vibrational sum frequency generation spectroscopy (BB-VSFG) is a promising tool toreveal information on the composition, orientation, interactions, and dynamics ofmolecules at surfaces and interfaces [2,3]. BB-VSFG is a nonlinear optical technique to obtain the vibrational spectrum of interfacial molecules, discriminating them from the bulk material. The combination of these two spectroscopic methods allows characterizing the interaction between the components of the liposomes, and those between liposomes and gold nanoparticles. Specifically, we have studied the effects of liposomal composition and investigated how the surface modification of nanoparticles influence and interact with the liposomes. Our data will provide insights for optimization of the ratio of components, amount of particles, etc. The obtained resultscan be useful in the field of nanomedicine. References [1] J. A. Webb, R. Bardhan, Nanoscale 6, 2502-2530 (2014). [2] H. F. Wang, W. Gan, L. Fu, Annu. Rev. Phys. Chem. 66, 189–216 (2015). [3] Z. Heiner, V. Petrov, M. Mero, APL Photonics 2, 066102 (2017).