Platform of Cellular Imaging and Cytometry (PIC2)
The PIC2 platform offers cellular analysis services based on two techniques:
1/ Flow cytometry and high-throughput cell sorting.
- Coordinator: Stefania Tolu (stefania.tolu@u-paris.fr)
2/ Total Internal Reflection Microscopy (TIRFM)
- Coordinator: Cécile Tourrel-Cuzin (cecile.tourrel-cuzin@u-paris.fr)
The plateau is open to all staff belonging to BFA research teams, but also to external research units and private companies.
Flow cytometry and cell sorting
PRINCIPLE
Flow cytometry is a technique for high-speed, quantitative analysis of a complex particle composition (cells, bacteria, parasites, beads…) on the basis of individual characteristics such as size, shape and granularity, and their fluorescence. Suspended particles pass, one by one, in front of one or more laser beams (question mark), and detectors pick up the signals emitted by each particle, such as :
- Forward scattered light (FSC), which provides information on particle size.
- 90-degree scattered light (Side Scatter – SSC), which provides information on particle shape, internal structure and granularity.
- Fluorescence signals (autofluorescence and fluorescence by labeling with antibodies or specific probes).
Cell sorting can be defined as the physical separation of cells or particles of interest from a heterogeneous population by electrostatic deflection of charged droplets. Cells are injected as a single stream through a nozzle into a continuous stream (jet). The jet is broken by the application of a vibration to produce drops at a precise point called the “break-off point”. The drops are electrically charged, pass between two highly charged deflection plates and are deflected to the side of the plate with the opposite polarity, before finally being collected.
TOOLS
The platform is equipped with a FACS Aria II (BD Biosciences), which includes :
➤ a temperature-adjustable injection chamber (4, 20, 37 or 42°C)
➤ 2 excitation lasers to detect up to 9 parameters
• Blue Laser (488 nm): 6 detectors (5 fluorochromes + granulosity)
• Red laser (633 nm): 2 detectors
➤ 5 nozzles (70, 85, 100, 130 µm): cell sorting of different sizes
➤ a tube sorting module for 1 to 4 populations of cells
➤ a module for sorting individual cell clones (ACDU module), in plate wells (24, 96 or 384 wells) or on a glass slide.
➤ An acquisition software (FACSDiVa™), which controls and adjusts the device and acquires data.
TECHNICAL APPLICATIONS
Cytometry can be used to carry out a variety of cellular analyses, including :
1/ analysis of cellular components (DNA, RNA, proteins, phenotyping, etc.)
2/ analysis of cellular functions (ion flow, pH modification, apoptosis, enzymatic activity, etc.)
3/ analysis of transfected cells (EGFP, PE, APC…)
All these measurements can be combined and, in some cases, accompanied by cell sorting.
Total Internal Reflection Microscopy (TIRFM)
PRINCIPLE
TIRF (Total Internal Reflection Fluorescence) microscopy, or evanescent field microscopy, is a form of fluorescence microscopy in which the excitation light is confined to a small area at the interface between the sample and the glass coverslip or culture vessel.
Many cellular processes involve the cell’s plasma membrane: the formation and stabilization of adhesion zones, membrane receptor trafficking, vesicle fusion or budding, or the recruitment of proteins to the plasma membrane. The study of these phenomena using TIRF microscopy enables observation with good axial resolution, acquisition speed and limited illumination cytotoxicity.
When a monochromatic polarized light beam (laser) illuminates an interface between two media of different refractive index (for example, the interface between a glass slide and a cell), part of the incident light is reflected at the interface and the other part of the light is refracted through the surface (A). Depending on the angle of incidence of the beam, all the light may be reflected. Under conditions of total reflection, one of the electromagnetic components of the light, the evanescent wave, propagates perpendicular to the interface with an intensity that decreases exponentially with distance from the interface (B).
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TOOLS
The platform includes a NIKON base mounted on an inverted fluorescence microscope (Ti-Eclipse). The microscope is equipped with a thermostated chamber (Life Imaging Series), enabling samples to be stored under physiological conditions.
This system is equipped with :
- Two 50mW Cobolt SLM (Single-Longitudinal Mode) lasers for fluorescence excitation at 488 nm and 560 nm
- Two Fluorescence cubes, one FITC cube (Excitation 488/10 nm; Dichroic mirror: 505 nm; Emission: 525/50 nm) and one TRITC cube (Excitation: 568/10 nm; Dichroic mirror: 565 nm; Emission: 610/60 nm).
- A multiband dichroic mirror
- Two objectives: a CFI Achromat LWD ADL 20X and a CFI Apochromat 60X TIRF O.N. 1.49 with oil for total reflection of the laser beam.
- A Photometrics camera EMCCD QuantEM 512SC (512×512 pixels; Roper Scientific)
- A control and acquisition software, MetaMorph version 7.5 (Molecular Devices).
APPLICATIONS
TIRF fluorescence microscopy enables the following analyses:
- Visualization of contact zones and cell adhesion to a substrate.
- Visualization of single molecules close to the membrane surface: membrane diffusion, protein/protein interactions, aggregations…
- Measure of protein binding rates to membrane receptors or adsorption to a surface.
- Follow-up of secretory granules on living cells, study of endocytosis and exocytosis mechanisms.
- Visualization of cytoskeleton morphology and dynamics.
Terms and conditions of use
CYTOMETER and CELL SORTER
📍Access
- Buffon Building A, 5th floor, room 532 A
📅 Appointment request
- mail to stefania.tolu@u-paris.fr (following feasibility study)
💰 Service rates
- Half-hourly rates for analysis and hourly rates for sorting.
| Hourly fees (€) | Analysis | Sorting |
| BFA Unit | 30€ | 50€ |
| UPC and CNRS laboratories | 33€ | 55€ |
| Public-sector laboratories | 42€ | 60€ |
| Private labs | 80€ | 170€ |
📓Publications et communications
- To ensure the visibility of the technical platform, users must cite the platform in the acknowledgements of publications including results obtained on the cytometer.
TIRF MICROSCOPE
📍Access
- Buffon Building A, 5th floor, room 532 A
📅 Appointment request
-
mail to cecile.tourrel-cuzin@u-paris.fr (following a feasibility study)
📓 Publications and Communications
- To ensure the visibility of the technical platform, users must cite the platform in the acknowledgements of publications including results obtained on the TIRF.