Flow cytometry (FCM) is a versatile technology, which allows for quantification of fluorescence and structural features of particles (most commonly cells). FCM analyzers provide rapid quantitative analysis of particles in suspension or soluble proteins from serum, fractioned cells, trypsinized cells or dissociated tissue. Researches and clinicians can obtain several statistics on a single cell and population level. Cell sorters can also analyze particles and physically separate cells of interest at high purity for downstream assays. A non-exhaustive list of different flow cytometry assays is listed below.
There are three major components to a flow cytometer: fluidics, optics and electronics. With exquisite pressure control and precise flow cell or nozzle design, the fluidics system hydrodynamicly focuses the sample and aligns the cells in single file. The cells then flow through the heart of the system, the interrogation point, where the fluidics meets the optics.
The optics are composed of both light excitation and light collection modules. At the interrogation point, one or several lasers are used to scan each cell one after the other to assess their physical and fluorescent parameters. The amount of light diffracted in line with the laser (Forward Scatter; FSC) provides an indication of size and laser diffraction at 90o (Side Scatter, SSC) provides an indication of cell complexity or granularity. In addition, cells can be labeled with reporter proteins, fluorescent dyes or fluorescently labeled antibodies, which selectively marks cells of interest. These sets of markers or color panels must be carefully chosen to be excited by the available excitation light source (lasers) and emit fluorescence at an emission wavelength of light that is distinctly collected by available band-pass filters.
The electronics components take advantage of photodiodes and ultra sensitive photomultiplier tubes (PMTs) to convert light, defined by the band-pass filters, into electronic pulses. These pulses are integrated, digitalized and sent to the acquisitions station, where the data can be interpreted.
The advantages of using FCM are that it is an extremely fast system and a relatively small quantity of sample is needed. Furthermore, in multiparameter FCM, several fluorescent parameters or colors are analyzed simultaneously. We can for example identify the phenotype and ascertain viability, vitality, proliferative capacity and cell cycle state of each cell. Therefore, since thousands of cells can be quickly analyzed, we identify extremely rare cell populations and also obtain population statistics with greater accuracy.
Flow Cytometry Applications (non-exhaustive list):
Detection of soluble molecules
DNA/Cell cycle analysis
Intracellular protein detection