Label-free quantitative biological imagingwith Lyncée Tec DHM®

Lipid droplets in adipocytes

Lyncée Tec supplies directly and through partners universities, industrial laboratories, and manufacturers with a complete range of Digital Holographic Microscopes (DHM®), application software, and accessories.

• Founded in 2003, in Lausanne, Switzerland

• Presence in 40 countries – A complete range of products

(direct and distribution network)– OEM partnerships

(semiconductor, photovoltaic, industrial metrology, life sciences)

• Lyncée provides turnkey solutions from sample handling to data analysis for:

– Optical Profilometry– Bio-imaging

Transmission DHM®

Reflection DHM®

Digital Holographic Camera

Lyncée Tec SAthe pioneer and leader in phase imaging

Our Life-sciences DHM® provides unmet advantages

Label-free non-invasive imaging technique (non-perturbing measurements)

Quantitative information about morphology and intracellular content

Millisecond to multi-days continuous recording

Digital Holographic Camera

To be attached to any existing microscope

Same specifications as DHM®

Non-perturbing measurements

Quantitative phase

Phase-fluorescence correlation

Additional channel for segmentation

Digital Holographic Camera specifications

DHC-S (Standard) DHC-P (Premium)

Acquisition rate 194 fps 75 fps

Field of view (10x) 0.6 * 0.6 mm 1.2 * 1.2 mm

Sensor 1 Mpixel sCMOS 4 Mpixel sCMOS

Fluorescence Sequential Simultaneous

• Magnification: from 5x to 100x

• Lateral resolution: MO dependent

• C-mount interface for high compatibility

Basis of Digital Holographic Imaging

Hologram

Numerical reconstruction+ analysis

On-the-fly Image analysis

DHM® quantitative phase signal:• morphological information• intracellular content• dynamics parameters• population metrics

Intensity image

Image acquisition

Microscope

Camera

Quantitativephase image

Software solutions

Acquisition and live analysis: Koala Advanced data processing: Cell Analysis Tool

Dedicated analysis workflows• End-point measurement• Time-lapse• Dose-response curve• Phase-fluorescence correlation• … and more

DHM®: Biological imaging / solutions4 key application categories

DHMsignal

4D tracking

Ion fluxes (optical patch

clamp)

Cell dynamics

Cell culture monitoring

Cell culture monitoring

Cytotoxicity

Live quantification of cytotoxicityHeLa cells treated with doxorubicin imaged continuously for 24 hours

Migration/proliferation

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0-1 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

T im e [h ]

Co

nfl

ue

nce

[%

](n

orm

ed

to

tim

e t

= 0

)

C t r l0 .3 n M C y to c h a la s in D1 0 µ M C y to c h a la s in D

Migration assayConfluence allows to quantify the effect of Cytochalasin D on the migration.Measured IC50 = 649 nM

Drug testing in 3D environment

HCT116 cells grown in 3D agar gel.Serial-dilution of doxorubicin. Image after 72h with at 10x

00 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

IC50Doxo_24h0.27

Doxo_48h0.065

Doxo_72h0.031

H C T 1 1 6 in 3 D a g a r g e l

D o x o r u b ic in [µ M ]

Dry

ma

ss [µ

g]

2 4 h

4 8 h

7 2 h

C tr l

Doxorubicin

Automatic reconstruction of a single plane is sufficient for quantification of cytotoxicity in 3D

Cell dynamics quantification

Cardiomyocytes beating

Beating profile quantificationNon-perturbing quantification of the beating (bpm, shape, etc.) of cardiomyocytes

Follow your cells in real time

Millisecond cell dynamics quantification

Intracellular organelles tracking

0 2 4 6 8 1 0

7 8

7 9

8 0

T im e [s ]

Avg

OPD

[n

m]

C tr l

C tr l

a fte

r 15

min

Isop r

e na l

ine

(10 0

nM

)

E -4 0

3 1 (1

0 0 n

M)

N ife

d ip i

n e (1

0 0 n

M)

F LP

6 41 7

6 (1

µM

)0

1 0

2 0

3 0

4 0

5 0

6 0

C a r d io m y o c y t e b e a tr a te m o d u la t io n (n = 3 )

Be

at

rate

[b

pm

]

Tracking: Refocusing capability(off-line digital propagation)

One hologram (single shot)from a single plane

Best focus(automatically found)

Range scanned: focus ± 140 µm

Bright-field Quantitative OPD image

z -p o s

[µm ]In

ten

sit

y

sp

ati

al

SD

b e s t fo c u s

No mechanical focusing-> faster acquisition

Digital positioning-> 3D localization

Bacteria 4D tracking

Tracking resolutionLateral = objective lateral resolutionAxial = depth of field/3(for 63x in air = ~0.5 µm in all dimensions)Transmission DHM, MO=63x

Bacteria 4D tracking

Transmission DHM, MO=63x

4D tracking

4D tracking of bacteria

Vibrio coralliilyticus bacteria tracks Non-perturbing quantification of the beating (bpm, shape, etc.) of cardiomyocytes

3D tracking in time with DHM®

Single acquisition per time point

Offline refocusing (typically at 50 im/s)

3D positioning and quantification at 194 fps

Multimodality

Fluorescence module

Simultaneous recording of DHM and FluoAllows to combine two modalities

Electrophysiology

Patch clamp setupRecord both the phase and current in the same sample

DHM® Fluorescence (fluo-4)

Added value with fluorescence modality

Combine multiple informations

China Agricultural University: Prof. Wang

Phaseimage

3D view

Combine multiple informations

Digital “flattening”of the water drop

Combine multiple informations

China Agricultural University: Prof. Wang

Confocal module:Z-stack reconstruction

HCS/HTS screeningDMSO DoxorubicinWhole plate snapshot

All physiological aspect of cells investigated simultaneously

384 well plate scanned in less than 11 minutes

Long time-lapse capability

Classify phenotypes

Conclusions

Label-free non-invasive imaging technique (non-perturbing measurements)

Quantitative information about morphology and intracellular content

Millisecond to multi-days continuous recording

Multimodality (software and hardware)

Contact informationLyncée Tec SAInnovation ParkBâtiment-ACH-1015 LausanneSwitzerlandwww.lynceetec.com

Tel.: +41 (0)21 693 02 20Fax: +41 (0)21 693 02 29info@lynceetec.com

Yves EmeryCEO

Benjamin RappazHead of life sciences applications

yves.emery@lynceetec.com

benjamin.rappaz@lynceetec.com

http://www.lynceetec.com/mailto:info@lynceetec.commailto:benjamin.rappaz@lynceetec.com

DHM® by Lyncée Tec scientific recognition1. Rappaz B, Moon I, Yi F, Javidi B, Marquet P, Turcatti G., Automated multi-parameter measurement of cardiomyocytes dynamics with digital holographic microscopy. Opt Express. 2015 May 18;23(10):13333-47.

2. P. Jourdain, F. Becq, S. Lengacher, C. Boinot, P. J. Magistretti and P. Marquet, "The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographicmicroscopy," Journal of Cell Science 127 (3), 546–556 (2014).

3. B. Rappaz, B. Breton, E. Shaffer and G. Turcatti, "Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening," Combinatorial Chemistry & High Throughput Screening 17(1), 80–88 (2014).

4. Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet and C. Depeursinge, "Marker-free phase nanoscopy," Nature Photonics 7 (2), 113–117 (2013).

5. N. Pavillon, J. Kühn, C. Moratal, P. Jourdain, C. Depeursinge, P. J. Magistretti, P. Marquet and J. Najbauer, "Early Cell Death Detection with Digital Holographic Microscopy," PLoS ONE 7 (1), e30912 (9 pages) (2012).

6. P. Jourdain, D. Boss, B. Rappaz, C. Moratal, M. - C. Hernandez, C. Depeursinge, P. J. Magistretti, P. Marquet and V. Ceña, "Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of ChlorideCurrent Associated to Activation of the Ligand-Gated Chloride Channel GABAA Receptor," PLoS ONE 7 (12), e51041 (10 pages) (2012).

7. Pascal Jourdain, Nicolas Pavillon, Corinne Moratal, Daniel Boss, Benjamin Rappaz, Christian Depeursinge, Pierre Marquet and Pierre J. Magistretti, "Determination of transmembrane water fluxes in neurons elicitedglutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic study," The Journal of Neuroscience 31 (33), 11846–11854 (2011).

8. J. Kühn, F. Montfort, T. Colomb, B. Rappaz, C. Moratal, N. Pavillon, P. Marquet and C. Depeursinge, "Submicrometer tomography of cells by multiple-wavelength digital holographic microscopy in reflection," Opt. Lett.34 (5), 653–655 (2009).

9. B. Rappaz, A. Barbul, A. Hoffmann, D. Boss, R. Korenstein, C. Depeursinge, P. J. Magistretti and P. Marquet, "Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy," Blood Cells,Molecules, and Diseases 42 (3), 228–232 (2009).

10. B. Rappaz, E. Cano, T. Colomb, J. Kuhn, C. Depeursinge, V. Simanis, P. J. Magistretti and P. Marquet, "Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographicmicroscopy," Journal of Biomedical Optics 14 (3), 034049 (5 pages) (2009).

11. B. Rappaz, A. Barbul, Y. Emery, R. Korenstein, C. Depeursinge, P. J. Magistretti and P. Marquet, "Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedancevolume analyzer," Cytometry Part A 73a (10), 895–903 (2008).

12. F. Charrière, N. Pavillon, T. Colomb, T. Heger, E. Mitchell, P. Marquet, B. Rappaz and C. Depeursinge, "Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba," Optics Express14 (16), 7005–7013 (2006).

13. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge and P. J. Magistretti, "Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy," OpticsExpress 13 (23), 9361–9373 (2005).

14. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb and C. Depeursinge, "Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cellswith subwavelength axial accuracy," Optics Letters 30 (5), 468–470 (2005).

And many others …

Whole range of options

DHM® specifications

1.25x to 100x microscope objectives (air and high NA oil)

1 nm accuracy in vertical axis

Acquisition rate: high SNR at 194 fps (optional up to 940 fps)

• Acquisition time per image: 0.5 ms

Cell dynamics and dry mass quantification

Cell cycle/dry mass

S. Pombe cell cycle monitoring

RBC physiological parameters

GABAA-R open30

µM

GAB

A

OPD = 312 nm

d: decrease (exit of water)nc: increase (concentration of cytoplasm)OPD: increase

Water follows Cl- fluxes -> changes in RI -> signal in DHM -> “optical electrode”

dnc

LargeOPD

Cl-

H2O

Optical electrophysiology

Phase = integration of current

1 min

6 nm0.2 nA

Increase of OPD =Exit of water

Inward current =Exit of negative ionsGABA (3 µM, 30s)

HEK cells with GABAA-R

Multimodal DHM®-Electrophysiology

Optical electrophysiology

Optical patch clamp

Cortical neurons glutamate responseIon-associated water movements is used to quantify the activity of ion channels and co-transporters

Optical recording of ions movements

Channels (GABA, glutamate, CFTR,…) and electroneutral co-transporteurs

No need to patch cells

No use of labeling agent

No phototoxicity

Simultaneous recording at each pixel of the field of view

Multi-site and parallel measurements

Label-free quantitative biological imaging�with Lyncée Tec DHM®Lyncée Tec SA�the pioneer and leader in phase imagingOur Life-sciences DHM® provides unmet advantagesDigital Holographic CameraDigital Holographic Camera specificationsBasis of Digital Holographic ImagingSoftware solutionsDHM®: Biological imaging / solutions�4 key application categoriesCell culture monitoringSlide Number 10Cell dynamics quantificationSlide Number 12Bacteria 4D trackingBacteria 4D tracking4D trackingMultimodalityAdded value with fluorescence modalityCombine multiple informationsCombine multiple informationsCombine multiple informationsHCS/HTS screeningConclusionsSlide Number 23Slide Number 24Whole range of optionsCell dynamics and dry mass quantificationOptical electrophysiologyOptical electrophysiology