Cell~vizioTM

Microscopic 

Resolution 

Macroscopic 

Resolution 

Cell~vizio TM 

Taking the Microscope into the Animal 

Invasive 

Intra-vital 

Imaging Systems 

Wide-field 

microscopy 

Two-photon 

Cell~vizio TM 

Functionalities Overview 

The Cell~vizio Solution 

Confocal 

microscopy 

Whole Body 


Bioluminescenc MRI 

e 

PET 

Fluorescenc 

X-ray e 

Noninvasiv 

e 

Minimally or non invasive 

microscopic imaging in situ 

in a living animal 

Microscopic 

Resolution 

Macroscopic 

Resolution 

Invasive 

> Cell~vizio Functionalities Overview 

> Wealth of Applications 

> Peripheral Nerve Imaging 

> Vascular Imaging 

> Snapshot of Various Applications 

Wide-field 

microscopy 

Current in vivo Imaging Practices 

Intra-vital 


Two-photon 

Confocal 

microscopy 

Whole Body 


Bioluminescenc MRI 

e 

PET 

Fluorescenc 

X-ray e 

Noninvasiv 

e 

Intra-vital Imaging 

> Microscopic resolution 

> Small FOV 

Whole Body Imaging 

> Macroscopic resolution 

> Large FOV 

Microscopic Resolution AND Minimally Invasive 

Fibered Fluorescence Imaging 

System 

Simple Contact 

Real Time 

QuickTime?and a 

YUV420 codec decompressor 

are needed to see this picture. 

YFP saphenous nerve. FOV 400 x 280 

µm 

1

Laser Scanning Unit 

• 488 nm coherent source 

• High-speed scanning system 

• Ultra-sensitive detector 

Cell~vizio’s Three Components 

ProFlex TM flexible microprobe 

• Tens of thousands of fiber optics 

• Specific high-precision connector 

• Custom miniature objective 

ImageCell TM software 

• Real time control 

• On-the-fly image processing 

• Quantitative capabilities 

Image Construction 




• 30 000 fibers 

• Point-by-point injection 

• Point-by-point detection 

• Optical section generated 

• Images formed at 12 

frames/second 

Fibered ProFlex to Access Virtually Anywhere 

> Proprietary connector 

> Micro-precision fiber to 

laser interface 

> Optimal laser injection 

> Automatically recognized 

by the LSU (i-button) 

> Bundle of tens of 

thousands of fiber 

optics 

> Monolithic bundle 

construction 

> Flexible and robust 

> Wide range of distal tips 

for various uses and 

applications 

> With or without optics 

> From 1.8 mm to 300 µm 


ProFlex 

ImageCell 


ProFlex 

ImageCell 

Miniaturized ProFlex Microprobes 

> Direct contact imaging by gliding ProFlex along tissue 




2

Miniaturized ProFlex Microprobes 

> Direct contact imaging by gliding ProFlex along tissue 

> Smallest diameter of 300 µm for penetration into soft tissue with minimal 

disturbance 

Simple Acquisition 

Controls 





ProFlex 

ImageCell 

Acquisition Tools 

Laser Power 

Control 

Movie 

Thumbnails 

LookUp Table 

Control 

> Maximum diameter of 1.8 

mm for confocal optical 

slicing with working 

distances between 20 and 

100 µm 

Confocal Optical Slicing 

Courtesy of Elisabeth Laemmel and Eric Vicaut, LEM, Paris, France 

z 

Quantificatio 

n 

ImageCell Versatile Software 




Calibration process for 

experimental repeatability 




Different horizontal planes visible 

with large vessel clearly above 

smaller one 

FOV 160x120 µm 

Controls system for proper 

image acquisition, treatment 

and viewing 

Analysis and Processing Tools 

Display 

Enhancing 

Comprehensive ROI 

Management 

5 Place Jules Janssen 

Enables post-acquisition 

image browsing and 

analysis 

Editing and 

Exporting 

3

External 

External 

Why Use the Cell~vizio TM? 

Versatile Use and Applicability 

Minimally 

Invasive 


Minimally 

Invasive 

Endoscopic 

Endoscopic 




Colonic crypts 

> Anyone can use the Cell~vizio, no particular training 

needed 

> Turn-key system requires no alignment or adjustments 

> Acquiring an image is as simple as placing the ProFlex 

in direct contact with a fluorescent organ 

> 

Immediate and Intuitive Use 

Hand-held ProFlex can be 

glided along fluorescent 

structures over long 

distances 

External 




Cornea epithelium 

External 

400 µm 





Minimally 

Invasive 


GFP Neurons in the 

striatum 

Minimally 

Invasive 




Isolated sensory fiber. FOV 400 x 

280µm 

GFP tumor cells 

Endoscopic 

Endoscopic 

4

Longitudinal Studies 

TABLETOP FLUORESCENCE MICROSCOPY 

• Sacrificed animal 

• Explanted and fixed nerve 

• One mouse per 

measurement 

• 50 minutes per measurement 

FIBERED CONFOCAL FLUORESCENCE MICROSCOPY 

• Live, anesthetized animal 

• In vivo and in situ imaging 

• Repeated measurement 

on the same mouse 

• 5 minutes per 

measurement 

Actual Case Study: Peripheral 

Nerve Imaging After Crush 

Injury 

> Three times fewer animals for three time-point study 

> 1/10 of time for each measurement 

> More relevant data with all measurements on same animal 

Morphometry and Signal Quantification 

> Basis for accurate and reliable 

morphometry and quantification 

> Integrated, real-time analytical tools 

> Quantification and comparison 

between sequences 

> Precise in-depth localization of 

objects thanks to confocality 

> Linear response over a large 

detection range 

Courtesy of E. Laemmel and E. Vicaut, Microcirculation Lab’, Hospital F. Widal, Paris, France 

Wealth of Applications 

Real Time Dynamic Sequences 

> Dynamic events captured at 12 frames per second 

> Stable and fluid sequences even with a hand-held 

ProFlex 

> Real time made possible by on-the-fly image 

processing 




Erythrocytes, stained ex vivo with FITC, 

going through a capillary. FOV 160 x 120 

µm 


Deep 

Brain 

Corne 

a 

Heart 






Ear 

Mesentery 

A World of Possibilities 

Lymph 

Node 

Liver 

Muscle 

Kidney 

Colon 

Bladder 

NMJ 

5

Excised tissue 

imaged with a 

fluorescence 

microscope 

In situ images 

acquired with the 

Cell~vizio 

Cancer Research 

Endoscopic In Vivo Histology of Rat Bladder 

Superficial 

prismatic 

cells 

Control cells 

Basal 

cells 

Published work: 

M.A. D'Hallewin, S. El Khatib, A. Leroux, L. Bezdetnaya, F. Guillemin 

“Endoscopic Confocal Flurorescence Microscopy of Normal and Tumor Bearing 

Rat Bladder” (2005)Journal of Urology - In press (August 2005) 

Peripheral Neuropathies 

Tumor 

cells 

Scale bar: 100 µm 

Subcutaneous 

Tumor 

Tumor Vasculature and Angiogenesis 

Applications: 

> Vasculature Characterization 

• Functional capillary density 

• Vessel diameter 

• Permeability 




Courtesy of Descartes Image, O. Clément, Necker School of Medicine, Paris, France 


> Angiogenesis 

• Endothelial cells recruitment 

• Anti-angiogenic therapies 

Observation of Aberrant Crypt Foci (ACF) 

Applications: 

> Colorectal cancer study 

> Aberrant Crypt Foci and adenoma characterization 

> Orthotopic tumor model observation 

> In vivo histology 

> Cytotoxic treatment monitoring 

Courtesy of Danijela Vignejevic, Sylvie Robine, Daniel Louvard, Institut Curie, Paris, 

France 





Image all Parts of Neuron in vivo and in situ 


TIFF (LZW) decompressor 








6

Vascularization 

Inject, Incise & Image 

Imaging is as easy as “1 2 3” 

1. Inject your dye IV 

2. Incise at the desired site 

3. Put ProFlex in contact with 

tissue 

Courtesy of Pr Vicaut, Microcirculation Lab, Hopital Fernand Widal, Paris, France 




Red blood cells with arrival of 

plasmatic dye 

FOV 400 x 280 µm 

Cell-endothelium interaction 




Rolling and adhering 

leukocytes 

Applications: 

> Leukocyte rolling 

>Recruitment of endothelial cells in neo-angiogenesis 

> Platelet aggregation 

> Atheromateous plaque formation 


Imaging from Vascular Networks to Single Cells 




Capillaries 




Cells in the 

blood stream 

Organ vascularization 




Vessels 

Courtesy of Ac Sinica, Taipei, Taiwan; CEA, Orsay, France; Hopital Fernand Widal, Paris, France 




Quantification of Vascular Parameters 

With ImageCell, measure: 

> Vessel diameter 

> Perfused capillary 

density 

> Permeability 

> Vasodynamics 

Published work: 

Laemmel, M. Genet, G. Le Goualher, A. Perchant, J.F. Le Gargasson, E. Vicaut 

« Fibered confocal fluoresence microscopy (Cell~vizio ｪ) facilitates extended imaging in the 

field of microcirculation » (2004) Journal of Vascular Research 41(5):400-411 

Other Applications 

8

Snapshot of Other Applications 

Ophthalmology Gene Expression 

Kidney 

Damaged cells of the cornea 

epithelium as revealed by topical 

application of fluorescein. 

YFP transfected muscular fibers 

from the mouse skeletal muscle. 

Pharmacokinetics Immune Responses 

Detection of a drug candidate at the 

membrane of liver hepatocytes after 

IV injection. 

CSFE labeled T cells detected in 

the inguinal lymph node after 

injection in the foot-pad. 

Kidney glomerulus in a beta-actin- 

GFP mouse. 

Courtesy of Rothschild Foundation, 

CEA-SHFJ, Animage (France), 

Stanford University (USA), CMU- 

Geneva Medical Research Center 

(Switzerland). 

FOV 400 x 280 µm 






Apoptotic bodies in a cornea graft as 

revealed by topical application of 

YoYo-1. 

Cells in a GFP-rice specimen. 

Snapshot of Other Applications 

Apoptosis In vivo Heart 

Pancreas 

Plants 

Cardiomyocytes in a ß-actin-GFP 

mouse. 

Excretion 

Excretion of small MW dextran in the 

kidney afetr IP injection. 

Glandular organization of the mouse 

pancreas as revealed by a Rhodamine 

123 topical staining. 

Courtesy of Rotschild Foundation, 

INRA Montpellier (France), 

Stanford University (USA), CMU- 

Geneva Medical Research Center 

(Switzerland), Hebrew University of 

Jerusalem (Israel) . 

FOV 400 x 280 µm 

Applying the Cell~vizio 

to 

Research in Peripheral Neuropathies 

Image all Parts of Neuron in vivo and in situ … Detect Immune Response and Vascularization 










> Local recruitment and 

infiltration of immune cells 

Capillaries after I.V. injection of FITC- 

Albumin 

FOV 400 x 280 µm 

Courtesy of C. Combadière, Cellular Immunology, Pitié-Salpétrière, and 

E. Laemmel, Microcirculation Lab’, Hospital F. Widal, Paris 

GFP T cells after adoptive transfer 

FOV 400 x 280 µm 

> Micro-architecture of the 

local vascular bed 

9

… For Wealth of Potential Applications 

> Peripheral nerve injuries 

> Diabetic neuropathies 

> Inflammatory demyelinating 

diseases 

> Drug-related neuropathies 

> Drug discovery for neurotrophic or 

neuroprotective molecules 

Easy Animal Preparation and Image Acquisition 

> Access to e.g. the saphenous 

nerve is only a small incision in 

the skin away 

> A simple contact with the 

fluorescent organ is enough to 

get real-time images 

> The hand-held ProFlex can be 

glided along fluorescent 

structures over long distances 

Courtesy of Igor Charvet and Paolo Meda, CMU, Geneva, Switzerland 

400 µm 




YFP saphenous nerve. FOV 400 x 280 

µm 

Monitoring Peripheral Nerve 

Injuries and Regeneration 

Follow Up of Whole Nerve’s Condition… 




D0, H0: Intact Saphenous 

Nerve 

… with a single fiber resolution 




D1 post crush: Degenerated 

Nerve 

D0, H1: Crushed 

Nerve 

D4 post crush: Regenerated 

Nerve 

Courtesy of Igor Charvet and Paolo Meda, Medical Research Center, Geneva, Switzerland 







Daily Quantification of Nerve Regrowth In vivo Screening of Drug Candidates 

Cell~vizio vs. Conventional Fluorescence Microscope 

Length of outgrowth (mm) 

9 

8 

7 

6 

5 

4 

3 

2 

Reliable measurements of nerve outgrowth with Cell~vizio imagin 


3 4 5 

Days after crush 




> Evaluate in vivo neurotrophic molecules and their 

effect on nerve repair after a trauma 

> Study neuroprotection in vivo and address drug- or 

disease-related neuropathies 

> Quicker and more relevant evaluation of 

neurotrophic/ neuroprotectant drug candidates 

Thy1-YFP mouse FOV 400 x 280 µm 

10

In vivo Detection of Nerve Endings 




Dendritic endings of a YFP nerve 

FOV 400 x 280 µm 





Motor endings of a YFP nerve 

FOV 400 x 280 µm 






Imaging from Vascular Networks to Single Cells 




Capillaries 




Cells in the 

blood stream 

Organ vascularization 

Vessels 




Courtesy of Ac Sinica, Taipei, Taiwan; CEA, Orsay, France; Hopital Fernand Widal, Paris, France 




Cell~vizio Benefits in Peripheral Nerve Studies 

> One instrument for all aspects of a pathology 

> Immediate and intuitive (real-time imaging, quick animal preparation) 

> Cost- and time- saving 

Example: to screen 100 candidate neurotrophic 

molecules on a 3 day follow-up 

300 mice 

250 hours 

100 mice 

25 hours 

> Longitudinal studies thanks to organ preservation and minimally 

invasive access to the organ 

Applying the Cell~vizio 

to 

Research on Vascularization 

Organ Vascularization within ProFlex Reach! 

Organ-Specific Patterns Vessels & Capillaries 

Liver 

Tongue 

Kidney 

Lung 

Courtesy of Ac. Sinica; CEA; Chang Gung University; Hopital Fernand Widal; Pasteur Institute. 

Mesentery Muscle 

Brain 

Ear 

12

Macromolecular leakage 

5, 11 and 25 min after 

histamine suffusion 

Extravasation 

Courtesy of Pr Vicaut, Microcirculation Lab, Hopital Fernand Widal, Paris, France 




Cell-Wall Interactions 

Leukocytes stained with 

Rhodamine6G, FITC-Albumin in the 

plasma 

FOV 160 x 120 µm 

Courtesy of Hopital Fernand Widal, Paris, France; UniMaas, Maastricht, Netherlands 

Extravasation 

1400 

1200 

1000 

800 

600 

400 

200 

0 

0 5 10 15 20 25 

Leukocytes stained with 

Rhodamine6G 

FOV 400 x 280 µm 

Time (mn) 




Observe, characterize and quantify the cell-wall interactions with minimal animal 

preparation! 

Meeting the Requirements for 

Vascularization Imaging 

Application Examples 

Tumoral Angiogenesis Made Visible 

> Minimal incision required to access subcutaneous tumors 

> High molecular weight dye to avoid leakage 

> The protocol can be applied for ischemia studies 

Courtesy of Anne-Carole Duconseille and Olivier Clément, Descartes Image, Small Animal 

Imaging Facility, Université Paris V, Paris, France 

> Access 




Injection of FITC-Dextran 500 kDa in a 

mouse with a subcutaneous PC3 tumor 

Requirements 

> Dynamic Events Recording 

> Study Vascular Development 

14

Cell~vizio Facilitates Access 

Conventional Lens: 

> X, Y & Z movement only 

> Diameter many mm’s 

• Major surgical disturbance 

• Difficult access 

• Constraints on experiments 

ProFlex: 

> X, Y, Z, theta & phi movement 

> Diameter < 1 mm 

• Minimal surgical disturbance 

• Easy access 

• Access to remote locations (ex. 

Kidneys) 

• New access possibilities (ex. Deep 

brain) 






Glomerulus of the Kidney 

Kidney glomerulus in a beta-actin-GFP mouse imaged with the ProFlex S-1500 in 

direct contact with the intact kidney. FOV 400 x 280 µm 

Courtesy of Christopher H. Contag and Tim Doyle, Stanford, 

CA 

Answer to Requirements 

> Dynamic Events: Fast and Slow Events 

Recording 

• Flowing red blood cells, rolling leukocytes => Real time recording 

• Vasoconstriction studies => Time lapse recording 

> Study Vascular Development 

• Longitudinal studies => Minimal invasiveness 

• Statistical analyses => Screening possibilities 

• Quantification needs => ImageCell tools 

Snapshot of Existing Applications 

with the Cell~vizio 

Tumor Capillaries 




Capillaries of a mouse subcutaneous prostate tumor stained by IV injection of FITC- 

Albumin. Circulating blood cells appear in negative contrast in the movie. Imaged 

using a ProFlex S-1500. FOV 400 x 300 µm 

Courtesy of Nathalie Faye, Laure Fournier and Olivier Clément, LRI, Faculté Necker,-EM 

Paris 

15

Dual Plane of Axial Resolution 

Microvasculature of mouse mesentery, showing two different planes of different 

microcirculation architecture. ProFlex S-1500. FOV 400 x 280 µm 

Courtesy of Mauna Kea Technologies, Paris, France 

Erythrocytes Circulating Through a Capillary 




Erythrocytes going through a capillary. Erythrocytes are stained ex-vivo with FITC 

while blood plasma is stained by FITC-Albumin. Imaged with a ProFlex HD-1800/80, 

2.5 fixed on a probe holder. FOV: 160 x 120 µm 

Courtesy of Elizabeth Laemmel, Eric Vicaut, Microcirculation Lab, Hopital Fernand Widal, 

Paris 

Colonic Mucosa 

Mouse colonic crypts stained with both Syto 13 (nuclear staining) and cresyl 

violet (cytoplasmic staning) ProFlex S-1500. FOV 400 x 280 µm 

Courtesy of Igor Charvet, Centre Medical Universitaire, Geneva, 

Switzerland 

Rolling Leukocytes 




Leukocytes rolling on a vessels wall. Leukocytes are stained with Rhodamine 6G 

while blood plasma is stained by FITC-Albumin. Imaged with a ProFlex HD-1800/80, 

2.5 fixed on a probe holder. FOV: 160 x 120 µm 


Paris 

Circulating Erythrocytes 




Circulation of erythrocytes in blood vessels. Erythrocytes are stained ex-vivo with 

FITC while blood plasma is stained by FITC-Albumin. Imaged with a ProFlex HD- 

1800/80, 2.5 fixed on a probe holder. FOV: 160 x 120 µm 


Paris 

In vivo detection of Aberrant Crypt Foci 




Movie acquired on a mouse treated with the carcinogen AOM. The colonoscopy 

was performed after instillation of acriflavine. ACF AOM-induced are clearly 

visible at the end of the movie. ProFlex S-1500. FOV 400 x 280 µm 

Courtesy of Danijela Vignjevic, Sylvie Robine, Daniel Louvard, Institut Curie, Paris France 

16

Orthotopic Visualization of a Colonic Tumor 

Visualization of a colonic AOM-induced tumor in a mouse. The colonoscopy was 

performed after instillation of acriflavine. ProFlex S-1500. FOV 400 x 280 µm 

Courtesy of Danijela Vignjevic, Sylvie Robine, Daniel Louvard, Institut Curie, Paris France 

Cornea Apoptosis 

Apoptotic bodies in a rejected cornea graft, marked with Yoyo1, imaged with a 

ProFlex S-1500 placed in direct contact with the cornea, with a totally non-invasive 

external access. FOV 400 x 280 µm 

Courtesy of Francine Behart-Cohen, U450, INSERM, Paris France 

Endoscopic Access to Bladder Umbrella Cells 

Umbrella cells of healthy urothelial mucosa after instillation of rhodamine 123 

(mitochondria marker), with cell nuclei appearing dark. Imaged with a ProFlex S- 

0650 in view of optical biopsies for early cancer detection. FOV 400 x 280 µm 

Text 

Courtesy of Samy Elkatib and M.A. D'Hallewin, Centre Alexis Vautrin, Nancy, France 

Cremaster Microvasculature 

Image showing mouse cremaster vessels stained with FITC-Albumin, injected 

intravenously, with vessels as small as 5 µm. Hand-held ProFlex S-1500. 

FOV 400 x 280 µm 


Endoscopic Access to Bladder Basal Cells 




Basal cells of healthy urothelial mucosa after instillation of rhodamine 123 

(mitochondria marker), with cell nuclei appearing dark. Imaged with a ProFlex S- 

0650 in view of optical biopsies for early cancer detection. FOV 400 x 280 µm 

Courtesy of Samy Elkhatib and M.A. D'Hallewin, Centre Alexis Vautrin, Nancy, France 

Endoscopic Access to Bladder Tumor Cells 




In vivo visualization of a tumor after instillation of rhodamine 123 (mitochondria 

marker), with cell nuclei appearing dark. Imaged with a ProFlex S-0650 in view of 

optical biopsies for early cancer detection. FOV 400 x 280 µm 

Courtesy of Samy Elkatib and M.A. D'Hallewin, Centre Alexis Vautrin, Nancy, France 

17

Homing T cells 

CFSE labeled T cells detected in the inguinal lymph node after injection in the 

foot-pad. FOV 400 x 280 µm 

Courtesy of Hamida Hammad and Bart Lambrecht, Erasmus MC, Rotterdam. 






18

Cell~vizioTM

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