April 20, 1985 Quantitative microscopy using 3D multicellular …filippopiccinini.altervista.org/PasswordSi/20160827... · 2016. 8. 23. · 1 Quantitative microscopy using 3D multicellular

1

Quantitative microscopy using 3D

multicellular spheroids:

generation, imaging, and analysis

Seoul, South Korea

27th August 2016 – 03rd September 2016

Eng. Filippo Piccinini, PhD

University of Bologna, Italy

Filippo Piccinini - details

First Name, Surname Filippo Piccinini

Place of birth Forlimpopoli, FC, Italy

Date of birth April 20, 1985

Bachelor degree Biomedical Engineer, University of Bologna, September 2004 - July 2007, score: 110/110 cum LAUDE

Master degree Biomedical Engineer, University of Bologna September 2007 - October 2009, score: 110/110 cum LAUDE

PhD degree European Doctorate in Information Technology

Email [email protected]

Italian mobile +39 3495000398

Website www.filippopiccinini.it

Current position

Post Doc Research Fellow, Advanced Research Centre on Electronic Systems (ARCES)

Computer Vision Group (CVG) University of Bologna, Italy

Supervisor Prof. Alessandro Bevilacqua

Where people typically think Italian researchers work Where my mother thinks I typically work

University of Bologna - Where I work Where I work - University of Bologna

2

Where I work - University of Bologna

UNIVERSITY OF

BOLOGNA

Computer Vision Group – Research interests

IMAGE PROCESSING AND ANALYSIS

Outdoor imaging Aerospace imaging Biomedical imaging

Filippo Piccinini, memberships

Italian Mesenchymal Stem Cell Group (GISM), www.gismonline.it, Founder Member

Italian Society of Biochemistry and Molecular Biology (SIB), www.biochimica.it

Italian National Bioengineering Group (GNB), www.bioing.it

European Association for Cancer Research (EACR), www.eacr.org

European Light Microscopy Initiative (ELMI), http://elmi.embl.org/home/

Network of European Bioimage Analysts (NEUBIAS), http://eubias.org/neubias/

Filippo Piccinini, main foreign collaborators

Prof. Peter Horvath, Biological Research Centre (BRC), Hungarian Academy of Sciences, Szeged, Hungary.

Prof. Kevin Smith, KTH Royal Institute of Technology, School of Computer Science and Communication, Stockholm, Sweden.

Dr. Gábor Csúcs, Light Microscopy and Screening Center, Swiss Federal Institute of Technology, ETH Zurich, Switzerland.

Prof. Valérie Vilgrain, Department of Radiology, University Beaujon Hospital, Paris-Clichy, France.

Dr. Vilja Pietiainen, Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.

Filippo Piccinini, developed software tools

CellTracker, for tracking cells cultured in vitro http://celltracker.website/

Advanced Cell Classifier, for classifying cells in high-content screening images http://www.cellclassifier.org/

MicroMos, for building a panorama, starting from a set of overlapping images

http://www.filippopiccinini.it/Mosaicing/index.html

AnaSP, software suite to segment brightfield images of multicellular spheroids http://sourceforge.net/projects/anasp/

ReViSP, for volume estimation and 3D rendering of multicellular spheroids http://sourceforge.net/projects/revisp/

CIDRE, for correcting the illumination field of microscopy images http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3323.html

3

Filippo Piccinini, publications

14) F. Piccinini, A. Tesei, A. Bevilacqua, Single-image based methods used for non-invasive volume estimation of cancer

spheroids: a practical assessing approach based on entry-level equipment. Computer Methods and Programs in Biomedicine,

advanced online publication, July 2016

13) C. Bellotti, S. Duchi, A. Bevilacqua, E. Lucarelli, F. Piccinini, Long term morphological characterization of Mesenchymal

Stromal Cells 3D spheroids built with a rapid method based on entry-level equipment. Cytotechnology, advanced online

publication, March 2016

12) M. Zanoni, F. Piccinini, C. Arienti, A. Zamagni, S. Santi, R. Polico, A. Bevilacqua, A. Tesei, 3D tumor spheroid models for in

vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific Reports, 6:

19103, January 2016

11) F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016

10) K. Smith, Y. Li, F. Piccinini, G. Csucs, C. Balazs, A. Bevilacqua, P. Horvath, CIDRE: an illumination-correction method for

optical microscopy. Nature Methods, 12(5): 404–406, May 2015

STATISTICS

Peer reviewed scientific articles: 22

- Journal publications: 14

- Conference proceedings: 8 First author publications: 10

Last author publications: 2

Total impact: 56.4050 IF Average impact: 4.3388 IF

Total number of citations: 170

H-index: 8

KEYWORDS

3D cell cultures

Light –sheet microscopy

Open-source software tools Mesenchymal stromal cells

Cell tracking

Cell classification

Filippo Piccinini, publications

09) F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular spheroids. Computer Methods and

Programs in Biomedicine, 119(1): 43–52, April 2015

08) F. Piccinini, A. Tesei, C. Arienti, A. Bevilacqua, Cancer multicellular spheroids: Volume assessment from a single 2D

projection. Computer Methods and Programs in Biomedicine, 118(2):95–106, February 2015

07) F. Piccinini, A. Tesei, G. Paganelli, W. Zoli, A. Bevilacqua, Improving reliability of live/dead cell counting through automated

image mosaicing. Computer Methods and Programs in Biomedicine, 117(3):448-463, December 2014

06) F. Piccinini, M. Pierini, E. Lucarelli, A. Bevilacqua, Semi-quantitative monitoring of confluence of adherent mesenchymal

stromal cells on calcium-phosphate granules by using widefield microscopy images. Journal of Materials Science: Materials in

Medicine, 25(10):2395-2410, October 2014

05) F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Automated image mosaics by non-automated light microscopes: the

MicroMos software tool. Journal of Microscopy, 252(3):226-250, December 2013

04) Z. Bulj, S. Duchi, A. Bevilacqua, A. Gherardi, B. Dozza, F. Piccinini, G. A. Mariani, E. Lucarelli, S. Giannini, D. Donati and S.

Marmiroli, Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells. International Journal of

Oncology, 42(1):118-126, January 2013

03) F. Piccinini, A. Tesei, W. Zoli and A. Bevilacqua, Extending the Universal Quality Index to assess N-image fusion in light

microscopy. International Journal of Bioelectromagnetism, 14(4):217-222, December 2012

02) F. Piccinini, A. Tesei, W. Zoli and A. Bevilacqua, Extended depth of focus in optical microscopy: assessment of existing

methods and a new proposal. Microscopy Research and Technique, 15(11):1582-1592, November 2012

01) F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Multi-image based method to correct vignetting effect in light

microscopy images. Journal of Microscopy, 248(1):6-22, October 2012

Outline

Spheroid generation

Pellet culture method

ReViSP and AnaSP

CellTracker

Advanced Cell Classifier

New project

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project

SPHEROID

“3D multicellular aggregate built in

vitro and used as a model for testing

drugs and radiotherapy treatments”

“In 1970 Sutherland proposed multicellular aggregates of "spherical" shape, called

spheroids, as a reliable 3D tumour model grown in vitro. These multicellular

aggregates morphologically resemble nodules seen in animal and human

carcinomas, and this is the reason behind the name spheroids.”

Cell culture: 3D VS 2D

3D CELL CULTURE 2D CELL CULTURE

Physiologic cell-cell contact Cell-cell contact only on the cell edges, and cells mostly in contact with plastic

Cells interact with extracellular matrix Cells contact extracellular matrix mostly on one surface

Diffusion gradient of drugs, gases, nutrients, and waste

No gradients present

Co-culture of multiple cells mimics microenvironment

Co-cultures unable to establish a microenvironment

MORE SIMILAR TO

IN VIVO TUMOURS

4

Spheroid placed in low-attachment multi-well plates

to test drug dosages or radiotherapy treatments

Plate preparation

Phantoms for linear accelerator

Round-bottom 96-well

low attachment plates

Oncology drug collection

Institute for Molecular Medicine Finland (FIMM) drug collection,

composed by 393 approved

and emerging investigational

oncology drugs.

PROBLEM

A cell-culture model used for testing drugs and radiotherapy treatments, in

replicates (for instance using several wells of a multi-well plate), must be

homogeneous and stable over time. Otherwise the data obtained are not reliable

and are dependent by the original status of the cells

HOW TO GENERATE HOMOGENEOUS AND

STABLE 3D SPHEROIDS?

DAY07, spherical shape DAY07, irregular shape

M. Zanoni, F. Piccinini, C. Arienti, A. Zamagni, S. Santi, R. Polico, A. Bevilacqua, A. Tesei, 3D tumor spheroid models for in vitro

therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific Reports, 6: 19103,

January 2016

Courtesy by 3DBiomatrix Courtesy by Synthecon Inc.

Common systems to build spheroids

Antigravity bioreactor Hanging drop plates

Courtesy by Hamilton

Magnetic levitator Pellet culture method

Comparison of systems to build spheroids

What do you want?

Parameters:

Number of homogeneous spheroids needed (High-content screening?)

Size of the necrotic core (Presence of a magnetic bead?)

Shape of the spheroids (Spherical? Irregular?)

Diameter of the spheroids (100µm or 1 mm?)

Parameters cell-line

dependent

Comparison of systems to build spheroids

What do you want?

Parameters:

Number of homogeneous spheroids needed (High-content screening?)

Size of the necrotic core (Presence of a magnetic bead?)

Shape of the spheroids (Spherical? Irregular?)

Diameter of the spheroids (100µm or 1 mm?)

Parameters

cell-line

dependent

Cell used:

A549 lung

cancer

TIME

REQUIRED

[days]

NO. CELL

REQUIRED

(×106)

EQUIVALENT DIAMETER

[µm]

(range, mean±SD, CV, n)

AMOUNT OF SPHERICAL

SPHEROIDS

(SI ≥ 0.90)

AMOUNT OF

LARGE SPHEROIDS

(diameter > 500 μm)

NASA

BIOREACTOR 15 40 500–1100, 897±98, 11.0, 192 HIGH HIGH

HANGING DROP

PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW

PELLET CULTURE

METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH

MAGNETIC

LEVITATION 7 0.5 200–500, 347±87, 25.1, 28 LOW LOW

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Correlation between viability and shape

We hypothesized that the 3D shape reflects a different general viability of

the spheroids. To better investigate

this correlation, we selected 30

spheroids of similar volumes (0.112 ±

0.013 mm3) but belonging to the spherical (n = 15; sphericity ≥ 0.90) or

non-spherical subtypes (n = 15;

sphericity < 0.90) to analyze how

different shapes influence the

metabolic state of spheroids. The data obtained from the luminescence

metabolic assay performed after one

week of culture showed a significantly

reduced viability of spherical spheroids

with respect to the irregular-shaped group (P = 0.045). This was probably

due to a reduced distance between

each cell and the culture medium

interface in the non-spherical subset,

leading to a wider zone of active cell proliferation

200 µm = maximum distance from border of the proliferating cells

Spherical shape

Ellipsoidal shape

Systems to build spheroids

What do you want?

High number of spheroids of same volume, large dimension, and spherical shape.

Cell used:

A549 lung

cancer

TIME

REQUIRED

[days]

NO. CELL

REQUIRED

(×106)

EQUIVALENT DIAMETER

[µm]


AMOUNT OF SPHERICAL

SPHEROIDS

(SI ≥ 0.90)

AMOUNT OF

LARGE SPHEROIDS


NASA


HANGING DROP

PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW

PELLET CULTURE

METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH

MAGNETIC



What do you want?


Cell used:

A549 lung

cancer

TIME

REQUIRED

[days]

NO. CELL

REQUIRED

(×106)

EQUIVALENT DIAMETER

[µm]


AMOUNT OF SPHERICAL

SPHEROIDS

(SI ≥ 0.90)

AMOUNT OF

LARGE SPHEROIDS


NASA


HANGING DROP

PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW

PELLET CULTURE

METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH

MAGNETIC



What do you want?


Cell used:

A549 lung

cancer

TIME

REQUIRED

[days]

NO. CELL

REQUIRED

(×106)

EQUIVALENT DIAMETER

[µm]


AMOUNT OF SPHERICAL

SPHEROIDS

(SI ≥ 0.90)

AMOUNT OF

LARGE SPHEROIDS


NASA


HANGING DROP

PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW

PELLET CULTURE

METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH

MAGNETIC


Conclusion – Morphological pre-selection

A morphological pre-selection of the spheroids, based on volume and sphericity is needed to obtain reliable data when using spheroids as in vitro models

F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular

spheroids. Computer Methods and Programs in Biomedicine, 119(1):2015.

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project

6

C. Bellotti, S. Duchi, A. Bevilacqua, E. Lucarelli, F. Piccinini, Long term morphological characterization of Mesenchymal Stromal

Cells 3D spheroids built with a rapid method based on entry-level equipment. Cytotechnology, advanced online publication,

March 2016 Pellet culture method

Cost-effective and extremely rapid method to generate MSC spheroids proposed by Johnstone et al. (In vitro chondrogenesis of bone marrow-derived mesenchymal

progenitor cells. Exp Cell Res, 1998).

It only requires a benchtop centrifuge and sterile polypropylene conical tubes

The generation efficiency is particularly high (one spheroid per tube) and the size of the spheroids can be tuned by controlling the number of cells seeded in the tubes.

Spheroid homogeneity

Cost of the system

Production efficiency

Tunable spheroid size


SPHEROIDS PRODUCTION

2.5×105 cells suspended in 0.5 ml DMEM-HG supplemented with 10% FBS and placed in a 1.5 mL polypropylene conical tube with a screw cap.

Aliquots were spun in a benchtop centrifuge at 500 g for 5 min.

Tubes incubated in humidified atmosphere at 37 °C with 5% CO2, with loosened caps to ensure adequate gas exchange.

After 72 h the pellets become compact spherical aggregates.

0.5 mm

Brightfield image acquisition

Plate preparation

Image acquisition

Original Corrected (*)

(*) K. Smith, Y. Li, F. Piccinini, et al., CIDRE: an illumination-correction

method for optical microscopy. Nature Methods, 12(5):2015

Illuminationcorrection

K. Smith, Y. Li, F. Piccinini, G. Csucs, C. Balazs, A. Bevilacqua, P. Horvath, CIDRE: an illumination-correction method for optical

microscopy. Nature Methods, 12(5): 404–406, May 2015

AnaSP: software suite to analyse several features

AnaSP (ANAlysis of SPheroid) SPHEROID SEGMENTATION

MORPHOLOGICAL FEATURES COMPUTATION

Software freely available at: http://sourceforge.net/p/anasp

F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular

spheroids. Computer Methods and Programs in Biomedicine, 119(1):2015.

7

Volume, Sphericity, and Jagging degree

25 MSC SPHEROIDS ANALYSED FOR A TWO-MONTH PERIOD

The intervals within the bounds represent µ ± 2σ

Volume, Sphericity, and Jagging degree

25 MSC SPHEROIDS ANALYSED FOR A TWO-MONTH PERIOD

The intervals within the bounds represent µ ± 2σ

External and internal architecture analysis

Cryostat Leica CM 1900

HISTOLOGICAL ANALYSIS CONFOCAL ANALYSIS

Nikon Eclipse Ti microscope equipped with an A1R confocal laser


LIGHT SHEET MICROSCOPE

Zeiss Light Sheet v2.1

PERFECTA3D and PROMEGA assays


HISTOLOGICAL ANALYSIS (H&E staining)

CONFOCAL ANALYSIS (Live&Dead assay)

Conclusion

The pellet culture method can be used efficiently to obtain homogenous

MSC spheroids with a high sphericity and a “smooth” surface

The MSC spheroids produced are morpho-biologically stable for at least

fifteen days after two weeks from generation

The two main outcomes of this work are showing that:

This is the first long-term analysis providing morphological and

biological data of MSC spheroids maintained in culture for two months

The pellet culture method can be used as a reference for laboratories

interested in easily obtaining stable homogeneous populations of MSC

spheroids without having to use specialized equipment

8

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project

F. Piccinini, A. Tesei, C. Arienti, A. Bevilacqua, Cancer multicellular spheroids: Volume assessment from a single 2D projection.

Computer Methods and Programs in Biomedicine, 118(2):95–106, February 2015

F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular spheroids. Computer Methods and Programs

in Biomedicine, 119(1): 43–52, April 2015

“Spheroid shape and volume are

relevant features for the data

reliability when spheroids are

used as an in vitro model”

State-of-the-art approaches for volume estimation

MODEL ASSUMPTIONS

Local spherical symmetry

Force of gravity

FORMULAS TYPICALLY USED

State-of-the-art approaches for volume estimation

MODEL ASSUMPTIONS

Local spherical symmetry

Force of gravity

FORMULAS TYPICALLY USED

SPHERE method

ELLIPSOID method

9

Methods from other application fields

SIERACKI method

M.E. Sieracki, C.L. Viles, K.L. Webb, “Algorithm to estimate cell

biovolume using image analyzed microscopy”, Cytometry Part A

10(5):551-557, 1989

ZEDER method

M. Zeder, E. Kohler, L. Zeder, J. Pernthaler, “A novel algorithm for

the determination of bacterial cell volumes that is unbiased by cell

morphology”, Microscopy and Microanalysis 17(5):799-809, 2011

Our approach

3D RECONSTRUCTION METHOD

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


DEPTH-OF-FOCUS RECONSTRUCTION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Input Output Input

Output

VIGNETTING CORRECTION

Our approach


SPHEROID SEGMENTATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


PROTUBERANCE SEGMENTATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


3D MAP CREATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

10

Our approach


INTERCONNECTING THE 3D PARTS

Single connection Parts interconnected with cylinders

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


SURFACE QUANTIZATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Analysis of a culture of spheroids

Mosaicing technique to acquire a high-detailed image of an entire well

F. Piccinini, A. Bevilacqua, E. Lucarelli, Automated image mosaics by non-automated light microscopes: the MicroMos software tool. Journal of Microscopy, 252(3):226–250, 2013.


Mosaicing technique to acquire a high-detailed image of an entire well

F. Piccinini, A. Bevilacqua, E. Lucarelli, Automated image mosaics by non-automated light microscopes: the MicroMos software tool. Journal of Microscopy, 252(3):226–250, 2013.

F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Automated image mosaics by non-automated light microscopes: the

MicroMos software tool. Journal of Microscopy, 252(3):226-250, December 2013


3D reconstruction of all the spheroids

Volume of the single spheroids

INPUT

OUTPUT

11

ReViSP software tool

Reconstruction and Visualization

from a Single Projection (ReViSP)

Microscope image GUI of ReViSP 3D mesh of the spheroid

ReViSP does not require any prior information except the binary mask of the spheroid to be reconstructed

ReViSP software tool

Reconstruction and Visualization

from a Single Projection (ReViSP)

Microscope image GUI of ReViSP 3D mesh of the spheroid

ReViSP does not require any prior information except the binary mask of the spheroid to be reconstructed

ReViSP live demo

Software freely available at: http://sourceforge.net/p/revisp

The ground truth volume of a spheroid

cannot be easily computed

problem

Ground truth volume

C: CONVERSION COEFFICIENT (voxels/mm3)

B: DENSITY

A: VOLUME BY OBJECT WEIGHTING

Sample object

Precision balance Graduated cylinder

Camera calibration grid

Synthetic objects (Group1)

SPHERICAL

OVOID

EIGHT

IRREGULAR

12

Synthetic objects (Group2 with protuberances)

ONE PROTUBERANCE TWO PROTUBERANCES THREE PROTUBERANCES

Volume assessment: Experimental results

Absolute percentage error, objects Group1

SPHERE ELLIPSOID SIERACKI ZEDER ReViSP

Spherical 0.69 1.99 1.09 0.74 1.26

Ovoid 23.97 8.15 2.45 2.01 2.08

Eight 21.91 33.12 4.34 4.96 1.65

Irregular 9.00 16.10 3.79 7.34 3.55

Average E% 13.89 14.84 2.92 3.76 2.14

Absolute percentage error, objects Group2

SPHERE ELLIPSOID SIERACKI ZEDER ReViSP

One protuberance 17.89 23.03 5.28 7.16 1.02

Two protuberances 11.79 6.45 13.77 9.35 5.89

Three protuberances 19.23 162.96 37.88 10.66 6.55

Average E% 16.30 64.15 18.98 9.06 4.49

Conclusion

Proved that common methods used to estimate volume of

spheroids are characterized by high errors.

Provided an approach conceived for 3D reconstruction and

visualization of a culture of spheroids.

Proposed a perspective gold standard method to estimate

the volume of a spheroid.

The main outcomes of ReViSP are:

Software freely available at: http://sourceforge.net/p/revisp


Rembrandt, 1629 Rembrandt, 1634 Rembrandt, 1640 Rembrandt, 1660

DAY08 DAY15 DAY22 DAY35 DAY01

15th July 1606

DAY08


AnaSP GUI

SPHEROID SEGMENTATION

MORPHOLOGICAL FEATURES COMPUTATION

Software freely available at: http://sourceforge.net/p/anasp


13

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project

F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016

F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016

CellTracker , freely available open-source software tool

http://celltracker.website/ ~1500 visitors a month!!!!!!!!!!!

CellTracker software ~200 download a month!!!!!!!!!!!!!!!

CellTracker GUI

CellTracker statistics

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project


Advanced Cell Classifier,

an open source software for automatically classifying

cells in high-content screening images

14


GRAPHICAL USER INTERFACE


CELL PHENOTYPES

Outline

Spheroid generation


ReViSP and AnaSP

CellTracker


New project

High-content screening in 3D

Generation of homogeneous and stable cancer spheroids to be used

as 3D tumour models.

Developments of automated image-processing methods and

machine learning approaches enabling cell phenotypic identification.

Validation of an approach to isolate single cells inside a 3D spheroid

by using a laser micro-dissector.

“[…] Recent improvements of computational capacity and automated

microscopy paved a new era in HCS, the 3D one […]”

SPECIFIC GOALS

OVERALL OBJECTIVE

develop computational and assay automation methods to perform 3D HCS


OVERVIEW

15


SPHEROID GENERATION

Anna Tesei, Oncology Research Hospital, Meldola, Italy

Laszlo Puskas, AVIDIN company, Szeged, Hungary

HIGH-CONTENT SCREENING

Leo Prince, OcellO company, Leiden, Netherlands

Main activities and collaborators

LIGHT-SHEET MICROSCOPY ANALYSES

Spartaco Santi, Digital Microscopy Center, Bologna, Italy

DRUG TESTING

Vilja Pietiainen, Institute for Molecular Medicine Finland, Helsinki, Finland

SOFTWARE DEVELOPMENT

Peter Horvath, Biological Research Center, Szeged, Hungary

Alessandro Bevilacqua, University of Bologna, Italy

SINGLE-CELL ISOLATION

Peter Horvath, Biological Research Center, Szeged, Hungary


Final dream?

Establishing a facility to perform 3D high-content screening analyses,

including spheroid generation, image acquisition, and cell classification

WHY AM I HERE IN SEOUL?

To see if there are opportunities for me,

new collaborations,

and to meet other researchers working in the field!

Advanced Research Center on Electronic Systems (ARCES), Computer Vision Group

(CVG), University of Bologna, Italy

Prof. Alessandro Bevilacqua

Dr. Alessandro Gherardi

Silvia Malavasi

Serena Baiocco

Biological Image Analysis and Machine Learning Group (BIOMAG), Biological

Research Center, Szeged, Hungary

Prof. Peter Horvath

Tamas Balassa

Abel Szkalisity

Csaba Molnar

Krisztian Koos

Arpad Balin

Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei

Tumori (IRST) IRCCS, Meldola, Italy.

Dr. Anna Tesei

Dr. Chiara Arienti

Michele Zanoni

Alice Zamagni

Orthopaedic Pathology and Osteoarticular Tissue Regeneration / Digital Microscopy

Center, Rizzoli Orthopaedic Institute, Italy

Dr. Enrico Lucarelli

Dr. Spartaco Santi

Dr. Barbara Dozza

Dr. Serena Duchi

THANK YOU

Dr. Chiara Bellotti

Dr. Elisa Martella

Other colleagues

Prof. Tullio Salmon Cinotti, Bologna, Italy

Prof. Kevin Smith, Stockholm, Sweden

Prof. Laszlo Puskas, Szeged, Hungary

Dr. Gabor Csucs, ETH Zurich, Switzerland

Dr. Vilja Pietiainen, Helsinki, Finland

Dr. Lassi Paavolainen, Helsinki, Finland

Acknowledgments THANK YOU

Eng. Filippo Piccinini PhD, www.filippopiccinini.it

Email: [email protected]

Mobile: +39 3495000398

Skype: filippo.piccinini85

Documents

April 20, 1985 Quantitative microscopy using 3D multicellular …filippopiccinini.altervista.org/PasswordSi/20160827... · 2016. 8. 23. · 1 Quantitative microscopy using 3D multicellular