Flow cytometry is a widely used method for analyzing, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume.
It allows simultaneous multi-parameter analysis of single cells.
It is commonly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules.
Regarding the fluidics, when a cell suspension is run through the cytometer, the sheath fluid is used to hydrodynamically focus the cell suspension through a small aperture. The tiny stream of fluid takes the cells past the laser light one cell at a time. Light scattered from the cells or particles is detected as they go through the laser beam. A detector in front of the light beam measures forward scatter (FS) and several detectors to the side measure side scatter (SS). Fluorescence detectors measure the fluorescence emitted from positively stained cells or particles. Cells or particles passing through the beam scatter light, which is detected as FS and SS. FS correlates with cell size and SS is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
A useful example of this is when running blood samples on the flow cytometer.
- Larger and more granular granulocyte cells produce a large population with high SS and FS.
- Monocytes are large cells, but not so granular, so these produce a separate population with high FS but lower SS.
- Smaller lymphocytes and lymphoblasts produce a separate population with less FS. They are not granular cells, so also have low SS.
Therefore, these cells can be separated into different populations based on their FS and SS alone.
As well as separating cells based on FS and SS, cells can also be separated by whether they express a particular protein. In this case, a fluorochrome is often used to stain the protein of interest. Fluorochromes (Thiazol orange for example) used for the detection of targeted proteins (RNA for example) emit light when excited by a laser with the corresponding excitation wavelength. These fluorescent stained cells or particles can be detected individually and counted.
Forward and side scattered light and fluorescence from stained cells are split into defined wavelengths and channeled by a set of filters and mirrors within the flow cytometer. The fluorescent light is filtered so that each sensor will detect fluorescence only at a specified wavelength. These sensors are called photomultiplying tubes (PMTs).