Transcript Titre - Accueil

Grappe Interdisciplinaire de Génoprotéomique Appliquée
Interdisciplinary Cluster in Applied Genoproteomics
What’s new at the GIGA Imaging and Flow
Cytometry Platform ?
Sandra ORMENESE & Gustavo MORAES (GIGA)
16/02/2012
Yannick MARRARI (BD Biosciences) & Rodolphe HAJJ (Servier)
GIGA-Research
GIGA-Flow cytometry
and Cell Imaging
Introduction
GIGA-Flow Cytometry and Cell Imaging

Created in April 2007 by gathering all the equipments (2 flow
cytometers, 1 laser microdissector and 1 confocal microscope) that belong
to different groups.
Different groups of researchers
19

Missions:
2007
25
55
53
2010
2011
37
2008 2009
☐
To train and advice the users
☐
To manage the equipment and to assure their maintenance
☐
☐
☐
To perform some special requests such as: cell imaging acquisitions,
images treatment/analysis in 3D & 4D, flow cytometry, laser
microdissection, cell sorting, rare tumor cell search and high content
screening
To help for funding applications
To develop new applications based on users needs and technological
progress
Platform Staff
GIGA-Flow Cytometry and Cell Imaging
Sandra Ormenese, PhD, Platform Manager



Management of all the equipment and their maintenance
Help for - funding applications
- Research program needing access to the platform
- New equipments acquisition
Development of new applications based on users needs and technological
progress
Gustavo Moraes, associate for Imaging Development


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Image treatment, analysis and interpretation (3D & 4D microscopy datasets)
High Content Screening
Cell Search
Raafat Stephan, technician

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Flow Cytometry training and advices
Flow cell sorting
Management of the daily tasks of the platform
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
☐
3 FACS analysers (up to 8 colors + FSC & SSC)
BD FACSCalibur 1 laser (488nm)
Calibur
BD FACSCalibur 2 lasers (488, 635 nm)
BD FACSCanto II (405, 488, 635 nm)
Canto
New cytometer analyser with loader that automates sample handling
with multitube racks and microtiter plates for walkaway operations.
Applications: cell cycle, cell ploïdy, cell proliferation, cell viability, measurement of a variety of
soluble and intracellular proteins including cytokines, chemokines, growth factors, and
phosphorylated cell signaling proteins, immunolabelling up to 8 colors…
☐
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
cloning)
Vantage
BD FACSVantage (2 ways, 405, 488, 635 nm)
Aria
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
☐
Different softwares for flow data analysis
ModFit, FlowJo, FCap, FACSDiva, CellQuest (Pro), FACSuite (soon), FlowCytomix Pro Software…
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
BD Fluorescence Spectrum Viewer
ATTENTION with the reporter genes
for red proteins as TagRFP
555
555
☐
584
584
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
Vantage
cloning)
BD FACSVantage (2 ways, 405, 488, 635 nm)
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
Aria
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
BD Fluorescence Spectrum Viewer
Yellow green laser
ATTENTION with the reporter genes
for red proteins as TagRFP
555
555
☐
584
584
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
Vantage
cloning)
BD FACSVantage (2 ways, 405, 488, 635 nm)
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
Aria
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
BD Fluorescence Spectrum Viewer
ALTERNATIVE: the TagRFP657
Yellow green laser
☐
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
Vantage
cloning)
BD FACSVantage (2 ways, 405, 488, 635 nm)
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
Aria
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
BD Fluorescence Spectrum Viewer
Yellow green laser
☐
ALTERNATIVE:
the TagRFP657
Red laser 635 nm
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
Vantage
cloning)
BD FACSVantage (2 ways, 405, 488, 635 nm)
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
Aria
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
BD Fluorescence Spectrum Viewer
Yellow green laser
ALTERNATIVE:
the TagRFP657
Red laser 635 nm
660/20 filter
☐
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
Vantage
cloning)
BD FACSVantage (2 ways, 405, 488, 635 nm)
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
Aria
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
☐
3 FACS analysers (up to 8 colors + FSC & SSC)
BD FACSCalibur 1 laser (488nm)
Calibur
BD FACSCalibur 2 lasers (488, 635 nm)
BD FACSCanto II (405, 488, 635 nm)
Canto
New cytometer analyser with loader that automates sample handling
with multitube racks and microtiter plates for walkaway operations.
Applications: cell cycle, cell ploïdy, cell proliferation, cell viability, measurement of a variety of
soluble and intracellular proteins including cytokines, chemokines, growth factors, and
phosphorylated cell signaling proteins, immunolabelling up to 8 colors…
☐
2 FACS sorters (in L2 labs, up to 8 colors + FSC & SCC, up to 4 sorting ways, single cell
cloning)
Vantage
BD FACSVantage (2 ways, 405, 488, 635 nm)
Aria
BD FACSAria II (4 ways, 405, 488, 635 nm,
aseptic sortings)
☐
Different softwares for flow data analysis
ModFit, FlowJo, FCap, FACSDiva, CellQuest (Pro), FACSuite (soon), FlowCytomix Pro Software…
Flow Cytometry
GIGA-Flow Cytometry and Cell Imaging
LUMINEX 100
It is a flow cytometry-based
instrument that integrates key
xMAP detection components
such as lasers, optics,
advanced fluidics and highspeed digital signal processors.
This system enables you to multiplex
(simultaneously measure) up to 100
analytes in a single microplate well, using
very small sample volumes (25 μL).
Fluorescence, phase contrast and
bright field microscopes
GIGA-Flow Cytometry and Cell Imaging
Bright field : sample illumination via
transmitted white light.
Phase contrast : shows differences in refractive index and in contrast.
Fluorescence : allows the detection of single molecules
using antibodies coupled to a fluorochrome
5 epifluorescence microscopes
Nikon E800 (upright)
Zeiss Axioplan (upright)
Olympus IX50 (inverted) (fits with ibidi slides & multiwell plates)
Olympus FSX100 All in one (inverted) (fits with ibidi slides & multiwell plates)
special plastic lens + stitching (3X3 or 5X5 mosaic montages) + short term time lapse imaging
Intravital Olympus Cell R (upright) with EMCCD camera
Confocal microscopy
GIGA-Flow Cytometry and Cell Imaging
4 confocal microscopes
☐
☐
☐
All equipped for time lapse
All equipped with spectral detection to select the ideal wavelength range
detection according to the fluorescence emission wavelength peaks
All equipped to develop FRAP, FRET & photoactivation
Olympus FV1000 (located in the B36)
Leica SP2
Leica SP5 AOBS (Acousto-Optical Beam Splitter)
Nikon A1R Hybrid resonnant scanner
Confocal microscopy
GIGA-Flow Cytometry and Cell Imaging
(at B36)


4 lasers (405, MultiAr 488, 559 and 635 nm)
2 spectral PMT, 1filter-based PMT, 1 PMT trans

10X, 20X, 20X LWD, 40X (oil), 60X (oil) objectives

DIC (Nomarski) for all objectives
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Motorized stage (multipoints time lapse)

It is NOT with an universal holder (ibidi slides not compatible)
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User friendly software, mainly for sequential scanning

Free FV1000 viewer can be downloaded and is easy to use
DIC
Confocal microscopy
GIGA-Flow Cytometry and Cell Imaging


3 lasers (MultiAr 488, 561 and 633 HeNe nm) : NO DAPI staining !
3 spectral PMT and 1 PMT trans

10X, 20X, 40X (oil) and 63X (oil) objectives

DIC (Nomarski) for 40X and 63X

It is NOT a motorized stage (multipoints time lapse
impossible)

It is NOT with an universal holder (only small Petri
dishes allowed for time lapse)

User friendly for playing with the PMT position
but NOT for sequential scanning

Free LCS viewer can be downloaded
DIC
Confocal microscopy
GIGA-Flow Cytometry and Cell Imaging
AOBS (Acousto-Optical Beam Splitter)
splits the excitation and emission light with a transparent crystal.

laser reflection and signal reception improved

less laser power needed

less photo-bleaching

up to 8 laser lines (simultaneously or in sequential)


4 lasers (405, MultiAr 488, 561 and 633 nm)
3 spectral PMT and 1 PMT trans

10X, 20X LWD and 63X LWD (glycerol) objectives

DIC (Nomarski) for all objectives

It is a motorized stage (multipoints time lapse, ATTENTION: NO CO2)

stitching

Equipped with an universal holder

User friendly software

Free LAS viewer can be downloaded
Confocal microscopy
GIGA-Flow Cytometry and Cell Imaging
Hybrid resonant scanner

Resonant scanner for high-speed imaging (230fps)

Non-resonant scanner for high resolution imaging up to 4096X4096 pixels

4 lasers (405, MultiAr 488, 561 and 639 nm)

32 spectral-based PMT (grating technology) and 1 PMT trans

4X, 10X, 20X, 20X LWD, 40X (oil), 40X LWD and 60X (oil) objectives

DIC (Nomarski) for all objectives exepted for the LWD

It is a motorized stage (multipoints time lapse)

PIEZO motorized Z-stage (high speed image acquisition in the Z direction)

stitching

It is with an universal holder
also for 96-well plates

Free NIS viewer downloadable & off-line advanced
treatment software
Laser microdissection
LMD 7000
GIGA-Flow Cytometry and Cell Imaging
to obtain homogenous, ultrapure samples from heterogenous starting material.
Laser microdissection uses a microscope to visualize individual cells or cell clusters in
bright field or in fluorescence modes.
Objectives: 5X, 10X, 20X, 40X, 63X & 150X
Regions of interest are selected by software, excised from the surrounding tissue by a
laser, and collected by gravity into specialized devices for analysis.
. Contact and contamination-free
. Transfer dissectates of any size or shape
Define Region
of Interest
Laser beam
along the cut line
Specimen
collection by
gravity
. Pool unlimited amounts of specimen
. Collect directy into medium
Laser microdissection
LMD 7000
GIGA-Flow Cytometry and Cell Imaging
To selectively and routinely analyze regions of interest down to single cells in the goal to
obtain results that are reproducible and specific.
Recommendations
GIGA-Flow Cytometry and Cell Imaging
Please:
 Respect the rules
 Pay attention to the
data management
 Feel free to ask
advices
 Have a look on the
brochure shelf
 Post your comments
on the web site
Image treatment and Analysis
GIGA-Flow Cytometry and Cell Imaging
Softwares: Bitplane Imaris, N.I.H ImageJ (FIJI), viewers (Leica, Olympus, Nikon), Attovision
Image treatment and Analysis
GIGA-Flow Cytometry and Cell Imaging
Imaris (BitPlane)

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Intuitif and semi-automatic 3D/4D image reconstruction, treatment and
analysis
Image treatment

Background correction/Signal optimization

Field of view edition (XYZT): 4D Crop, Extended Projection, ...
Image Analysis

Cell counting/Migration analysis
Image treatment and Analysis
GIGA-Flow Cytometry and Cell Imaging
Imaris (BitPlane)



Intuitif and semi-automatic 3D/4D image reconstruction, treatment and
analysis
Image treatment

Background correction/Signal optimization

Field of view edition (XYZT): 4D Crop, Extended Projection, ...
Image Analysis

Cell counting/Migration analysis

Neurite tracking
Image treatment and Analysis
GIGA-Flow Cytometry and Cell Imaging
Imaris (BitPlane)



Intuitif and semi-automatic 3D/4D image reconstruction, treatment and
analysis
Image treatment

Background correction/Signal optimization

Field of view edition (XYZT): 4D Crop, Extended Projection, ...
Image Analysis

Cell counting/Migration analysis

Neurite tracking

Volume measurement

Colocalization analysis (quantification)
Image treatment and Analysis
GIGA-Flow Cytometry and Cell Imaging
Imaris (BitPlane)

Movie maker (Quicktime, AVI, MP4, TIFF series, ...)

Statistics visualization and export (.csv)
Cell Search
GIGA-Flow Cytometry and Cell Imaging
Cell Search System (Veridex/J&J)

Circulating Tumor Cells (CTC) Isolation and Analysis

Overall Survivor and Progression Free Survival predictor

Patient prognostics and therapy modulation
Cell Search
GIGA-Flow Cytometry and Cell Imaging
Cell Search System (Veridex/J&J)

Circulating Tumor Cells (CTC) Isolation and Analysis

Overall Survivor and Progression Free Survival predictor

Patient prognostics and therapy modulation

Capable to isolate down to 1 single CTC in 7.5mL of blood

Standardized and automated procedure

FDA approved
Cell Search
GIGA-Flow Cytometry and Cell Imaging

CellTracks AutoPrep
☐
CellSave Tubes
• 92 hours conservation
☐
CTC Kit
☐
Magnest cartridge
Cell Search
GIGA-Flow Cytometry and Cell Imaging

CellTracks Analyser
☐
CTC detection
☐
Leukocytes differentiation
Cell Search
GIGA-Flow Cytometry and Cell Imaging
Cell Search System (Veridex/J&J)

Circulating Tumor Cells (CTC) Isolation and Analysis

Overall Survivor and Progression Free Survival predictor

Patient prognostics and therapy modulation

Capable to isolate down to 1 single CTC in 7.5mL of blood

Standardized and automated procedure


FDA approved
Ready-to-use Kits for In Vitro Diagnostics or Research

Research kits:

Endothelial Cells Kit/ Melanoma Cells Kit
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Profile kit

Tumor Phenotyping (HER2/neu, EGFr) Kit
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Mouse/rat Kit
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User-defined labeling Kit (CXC)
High-Content Screening
GIGA-Flow Cytometry and Cell Imaging
BD Pathway 855

Main subject of this meeting

Features
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Automated procedure for large sampling

Fast multi-point acquisition

Time consuming gain
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Less phototoxic (spinning disk)
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Fluidics handling station (automatic)

Image and data analysis

GIGA Imaging Platform workflow
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Invited experts

Dr. Yannick MARRARI


Application specialist – BD Biosciences Europe
Dr. Rodolphe HAJJ

HCA Imagery facility manager – Institut de recherches Servier - France