La Bio-Imagerie En fluorescence pour l'Exploration Tissulaire Et cellulaire,  du nouveau sur la plateforme APEX

La Bio-Imagerie En fluorescence pour l'Exploration Tissulaire Et cellulaire, du nouveau sur la plateforme APEX

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Description: This presentation is about La Bio-Imagerie En fluorescence pour l'Exploration Tissulaire Et cellulaire, du nouveau sur la plateforme APEX. Journée scientifique de l’axe Bio-imagerie biogenouest 4-02-14. Synergy between two fieds of expertise.

Venerinary pathology, Autopsy Lesionnal investigation, Diagnosis, Histomorphometry. Fluorescence bio imaging celluler typing, Co-labeling, 3D imaging, Spectral imaging, Image analysis. alidating animal models.

Animal models of chikungunya disease for gaining insight into persisting arthralgia physiopathogeny Spontaneous Induced (chirurgical, targeted mutagenesis) from tissue and cell tracking to physiopathogeny Multilabelling, Highly sensitive detection, Ecotoxicology, Whole-organism studies, Preclinical testing Efficacy and safety evaluation. Integrated offer for tissue phenotyping. APEX & other open platforms in Europe.

Expertise in pathology study of morphological changes (lesions) of tissues and organs associated to diseases. A veterinary pathologist should be usefull to identify normal versus abnormal appearance significant versus unsignificant lesion interspecies variations uncommon lesions primary lesions versus secundary lesions. Expertise in tissue exploration by fluorescence bio-imaging, A spectral confocal microscope since november 2013 at APEX.

A spectral confocal microscope, a powerful tool to separate specific signal from auto fluorescence.

 
Author: Laurence Dubreil, Thibaut Larcher  (Fellow) | Visits: 2892 | Page Views: 3586
Domain:  Medicine Category: Veterinary Subcategory: Pathology 
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Contents:
La Bio-imagerie en fluorescence pour
l'exploration tissulaire et cellulaire,
du nouveau sur la plateforme APEX

APEX
Laurence Dubreil / Thibaut Larcher

Journée scientifique de l’axe
Bio-imagerie biogenouest 4-02-14

12/ 6/ 2013

UMR703 PAnTher
Physiopathologie Animale et bioThérapies du
muscle et du système nerveux central
#15 permanent staff members
# 4 PhD students
Headed by M-A. Colle and K. Rouger

Cellular
interactions
Cell tracking

Cell therapy of
muscle tissue
1 EC (5%)
1 sec. (5%)
1 TR (30%)

Pathogen
tracking

Gene therapy
of central
nervous system

1 EC (10%)
1 AI (10%)
3 ingénieurs (80-70-50%)
1 assistant ing. (50%)
2 techniciens recherche (100%)

Transduction
profiles

APEX

Biological agent
biodistribution

.02
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

APEX

Autopsy
Lesionnal investigation
Diagnosis
Histomorphometry

Cellular typing
Co-labeling
3D imaging
Spectral imaging
Image analysis

Thibaut.larcher@nantes.inra.fr

Laurence.dubreil@oniris-nantes.f

.03

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 3 / 2014
02

Scientific fields
One World, One Medicine, One Health

Validating animal models

Human

Mouse

Animal models of chikungunya disease for gaining
insight into persisting arthralgia physiopathogeny

 Spontaneous
 Induced (chirurgical, targeted mutagenesis)

From tissue and cell tracking to physiopathogeny
 Multilabelling
 Highly sensitive detection

Ecotoxicology
 Whole-organism studies

Preclinical testing
 Efficacy and safety evaluation

scAAV9 intracisternal delivery to the central
nervous system of a feline model of moto-neuron
disease

.04
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Integrated offer for tissue phenotyping
Tissue sampling
Tissue trimming





Vibrating cutting device
Cryostat
Microtome
Unltramicrotom

>1100 blocks and >2200 slides per year

Topographic staining
Immunostaining and histoenzymology
In situ hybridization

Fluorescence bio-imaging
Histomorphometry

.05
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

APEX & other open platforms in Europe
With at least 1 ECVP/ACVP diplomate

CReSA (Barcelona)
Finnish Centre for Laboratory Animal
Pathology (Helsinki)

Human histopathology and animal models
(Pasteur Institute, Paris)
MIRCen (INSERM-CEA, Fontenay-aux-Roses)

Moredun (Penicuik)

Mouse & Animal Pathology Lab (Milan)
VIB – Center for the Biology of Disease
(Louvain)

Field of expertise:
Animal model or Target species

.06
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Expertise in pathology, what’s for ?
(Anatomical) Pathology: study of morphological changes (lesions) of tissues and organs
associated to diseases
A medical speciality (recognised training program for veternarians of 3 to 3.5 years in
France)

A veterinary pathologist should be usefull to identify…






normal versus abnormal appearance
significant versus unsignificant lesion
interspecies variations
uncommon lesions
primary lesions versus secundary lesions

.07
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Expertise in tissue exploration by fluorescence
bio-imaging, what’s for?
Motorized wide-field
fluorescence
microscopy, 80i Nikon

Cell and tissue phenotyping



Multi-labelling
Colocalization

Cell tracking
• Multi-labelling
• 3D imaging

Confocal laser
scanning
microscope
Nikon C1

Tracking of molecules


Multi-labelling




Spectral imaging
Online linear unmixing

GFP + Noise

GFP

Noise

Spectral confocal microscope
LSM 780 Zeiss with GaAsps detectors

.08

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 8 / 2014
02

Cellular and tissular phenotyping of SNC
Origin of demand = U1089 Inserm Nantes, gene therapy of SNC

APEX offers :
Training : tissu sampling, tissu triming, multilabeling, fluorescence bio-imaging
Confocal microscopy, Morphometry, Neuropathology.
Bucher and al, Gene Therapy, January 2014 accepted

.09

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 9 / 2014
02

Embryon 3D imaging and image analysis
Origin of demand : sécurité sanitaire des biotechnologies de la reproduction, Oniris

APEX offers :
Development for embryon imaging
Training : multilabeling, image analysis
Confocal microscopy, 3D imaging
Moreno D and al., Reproduction, Fertility and Development 26(1): 154. January 2014.

.010

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

10
4/ 02 / 2014

Molecular tracking in rat liver
Origin of demand : private company
Injection substance
X-rhodamine
t15min, t2h, t4h…

Tissu sampling, liver, spleen, lung
Fluorescence
bio-imaging

Veterinary pathology

cryosection

paraffin embedding, sectioning

multilabeling

HES staining

Confocal microscopy
Image analysis

Lesional
investigation

Report writing
APEX offers: Veterinary patholgy expertise and tissular exploration by fluorescence bio-imaging
.011

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

11
4/ 02 / 2014

A spectral confocal microscope since november
2013 at APEX

-

6 raies laser : 405, 458, 488, 514, 561, 633 nm
32 détecteurs GaAsps spectraux et 2 PMT
1 TPMT/DIC
Acquisition confocale FRAP
Multilabeling, spectral imaging, spectral unmixing

.012

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

12
4/ 02 / 2014

A spectral confocal microscope, a powerful tool
to separate specific signal from autofluorescence
1)Example : spinal cord
Confocal channel mode

? ?

Lambda acquisition
from tissue control

Online fingerprinting

?

?

2)Example : aorta

__ GFP spectra
___ Autofluorescence

Neurone GFP+
Autofluorescence

Spectral image

autofluorescence spectra
of elastic fibers

.013
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

New offers in tissue and cell exploration by
fluorescence bio-imaging in 2014
Membrane trafficking, TIRF imaging




TIRF

EPI

High resolution at the membrane level (80 nm)
Trafficking of molecules and organelles to and from the cellular
membrane
Binding of ligands to their membrane-bound receptors

Phenotyping of living tissues, Biphotonic imaging




Deeper tissue penetration and low phototoxicity by using infrared
laser beam
Intravital microscopy
Imaging of second harmonic generation signals

.014

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

14
4/ 02 / 2014

Total Internal reflection fluorescence microscope
TIRF : an evanescent wave to selectively illuminate and
excite fluorophores in the region immediatly adajacent to
the glass water interface. Resolution 80-100 nm

Digital EMCCD Camera
objX63, NA 1,46

.015

APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

15
4/ 02 / 2014

Biphotonic imaging, what’s for in tissular and
Biphotonic imaging
cellular exploration in vivo?
Principe
2Photon excitation

1Photon excitation

Excitation
488 nm

Fluorescence
emission

Excitation
976 nm

Fluorescence
emission

Major Inputs of 2Photon excitation:

Femtosecond laser ( infrared laser beam 680-1080 nm)

-

Deeper tissu penetration
Reduction of the photoxicity
Imaging of living tissu
Imaging of second harmonic signals

.016
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Biphotonic imaging, what’s for in tissular and
cellular exploration?
Major Input : deeper tissu penetration (2X with biphotonic
microscopy)
Scaled brain GFP expression

A1RPM, Nikon, MiFoBio 2012

Second harmonic generation signal
(l/2), molecular conformation and
orientation (fibrillar collagen).
sciatic nerve from a mice

Collagen matrix : second harmonic
generation

L. Dubreil, APEX UMR 703

.017
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Biphotonic imaging, what’s for in tissular and
cellular exploration?
Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9459-64

Major input : Imaging of living tissu

Adult transgenic Thy1GFP-M mice
Rhodamin B isothiocyanate-Dextran

18

Biphotonic imaging, what’s for in tissular and
cellular exploration?
Imaging of second harmonic generation signals : A tool to explore fibrosis (collagene type I)

(1) EA

4427 SeRAIC - INSERM U522, IFR 140, Université de Rennes 1, Hôpital Pontchaillou, 35033 Rennes, FRANCE
de Physique de Rennes, UMR UR1-CNRS 6251, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes, FRANCE
(3) Liver Department Hôpital Pontchaillou, 35033 Rennes, FRANCE
(4) Department of Pathology, Hôpital Pontchaillou, 35033 Rennes, FRANCE
(5) EA SeRAIC - INSERM U620, IFR 140, Université de Rennes 1, 35043 Rennes, FRANCE
(6) Laboratoire de Spectrométrie et Optique Laser, EA 938, Université de Bretagne Occidentale, 29238 Brest, FRANCE
(7) Université Européenne de Bretagne, 5 Boulevard Laënnec, 35000 Rennes, FRANCE
(2) Institut

We first validated the selectivity of SHG signals for fibrotic deposits (fibrillar
collagen type I and III) in human liver by establishing a strong correlation between
SHG and immunochemistry assays.
Both frozen fixed or paraffin embedded tissues can be used
SHG scoring gives the opportunity to quantify fibrosis regression
in response to an antifibrotic treatment for validating experimental
protocols especially for cirrhotic patients
SHG imaging and fibrillar collagen scoring will be applicable
to endoscopic systems by using optic fibers coupled
with multiphoton microscopy, thus leading to a less invasive
procedure for fibrosis evaluation

19

Types of offers
Training and support

GFAP

 Multi-labelling (IHC/ISH…)
 Tracking
 3D imaging

Training






Beta-galactosidase
Dystrophine
Laminine

Development

Tissue sampling of lab animals
Lesion recognition and description
Fluorescence bio-imaging
Histomorphometry
Workshops (Imaris, Perkin Elmer)

10 individual training sessions since 2010 and 6 seminars for research actors
A mean of 25 days per year dedicated to training

.021
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014

Interactions with other plate-forms
APEX

LTG

Fluorescence Bioimaging,
Veterinary Pathology

Viral vector production
for gene therapy

NeurATRis
Infrastructure Nationale
En Biologie Santé
Biothérapie en
neurosciences

Collaborations

P. Moullier

Consortium CEA

Training and seminaries, exchanges of knowledges

Micromorph
Histology plateform

BIBS
Microscopy, MCBL, MET,
AFM

CEISAM
Development of
fluorescent probes

MicroPiCell

Myriam Robard

Microscopy, MCBL, HCS,
Video microscopy

B. Bouchet - C. Gaillard
E. Ishow

P. Hulin - S. Nedellec

21
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

.021

4/ 02 / 2014

http://www6.inra.fr/anatomie_pathologique_sante_animale

.022
APEX / Expertise in Veterinary Pathology and fluorescence bioimaging

4/ 02 / 2014