Flow cytometry principle
During the last decade, flow cytometry has become a very powerful technique and impacted a wide range of fields from basic cell biology to genetics, immunology, molecular biology, microbiology and environmental science. The principle of flow cytometry can be easily understood from its definition : measurement of the optical properties of cells (cyto-) flowing at the very centre of a liquid sheath as they are individually intercepted by a light source focused at a very small volume. Particles (i.e. cells) are interrogated in a very short time (2 to 10 μs) during which several optical signals are detected and collected, mainly light scatter and fluorescence emissions in the visible spectrum.
Signals coming from the cells are photons either from the laser and scattered by the cells, either produced by the cell fluorescence. Several optical filters are used to separate the different photons according to their wavelengths and drive them to the photodetectors (most often photodiodes for light scatter and photomultiplier tubes for fluorescences). Data are then displayed and interpreted in order to discriminate clusters of particles based on statistical analyses of the parameters collected.
Why is flow cytometry so popular among microbiologists ?
Flow cytometry analyses are very fast (up to several thousands cells per second). A very large amount of cells can thus be analyzed per sample (up to 10 millions !), and the statistical results are obviously representative of the population. That also allows analyzing more samples, a key issue in spatial or temporal studies. Using these statistical analyses, it is possible to electronically separate these populations and identify them using multivariate analytical techniques. Flow cytometers can nowadays readily analyze single cells at rates up to 100,000 per second. Flow cytometry provides multiparametric analyses at the single cell level (several scatters and fluorescences). The data are quantitative data and can be correlated to other biochemical data. Moreover data are acquired in real time. The size class distribution of microorganisms can be assessed from the scattered light and the analysis also provides the cell abundance. The presence of unique identification markers in the cells allows separating cell clusters. These markers can be natural in the case of auto-fluorescent cells (example : natural fluorescent pigments) or induced by adding fluorescent dyes (fluorochromes) that target particular cell component or activity. The last, but not the least, some flow cytometers called “sorters” are able to physically separate the clusters of interest. Upon sorting, cells can be collected in a tube, a Petri dish, a 96-well plate, etc. In most cases sorting does not affect viability and post-cultures are possible. Sorting is also very valuable for post-analyses (molecular biology).
Unfortunately flow cytometers are very expensive instruments, very often out of reach for laboratories. Moreover they require highly trained people. This is why the “Plateforme Régionale de Cytométrie pour la Microbiologie” PRECYM has been created in 2005.
The Regional Flow Cytometry Platform for Microbiology (PRECYM)
Aware of the potential of this tool, the three laboratories of the COM (LMGEM UMR 6117, LOB UMR 6535, and DIMAR UMR 6540), as well as two Research Units of the IRD (UR 99 Cyroco and UR 103 CAMELIA), and the Laboratoire de Chimie Bactérienne (LCB UPR CNRS 9043) organized a consortium with the aim of acquiring a flow cytometer cell sorter (a MoFlo from DAKO, Dk). The Regional Flow Cytometry Platform PRECYM was created in 2005 and centralizes this equipment and know-how and makes them available to other units/institutes. PRECYM also allows the optimal technical, economic, and scientific exploitations of the instruments. PRECYM is also involved in the R&D and in the technological survey, as well as the research valorisation.