What Is Flow Cytometry?

Flow cytometry and microscopy share a great deal in common; at their root, both techniques allow us to investigate biological molecules and biochemical events using fluorescent probes (as well as non-fluorescent measurements). For example, one can employ fluorescently labeled antibodies to investigate proteins of interest or use “functional state” fluorochromes as a readout of pH, calcium, reactive oxygen species, cell viability, DNA content and many other critical biological events. However, flow cytometry and microscopy also have several differences which will dictate which method is best suited to your project (or perhaps both should be utilized in conjunction). 

The “strength” of flow cytometry comes from its central premise of analyzing a suspension of single cells very rapidly and generating highly quantitative fluorescence information. This allows us to look at sub-populations, rare cell events, cellular differentiation, etc. in a matter of seconds or minutes. Typically 10,000 cells can be analyzed for over 10 parameters: 10 fluorescence markers, “size” (parameter is related to refractive index and other variables as well), intracellular granularity, etc. This can all be done in less than one minute and we can determine how much of each fluorescent marker was present in the sample. In particular, this level of throughput and quantitation are critical in assessing rare cell populations (progenitor cells, stem cells, circulating tumor cells, etc).

Quite importantly, flow cytometry gives information on each and every cell unlike other methods that simply provide an average. If you created an “average” of all the people at a hockey game, it would look nothing like the critical sub-populations (the hockey players themselves, the very rich people sitting in the gold seats or the die-hard fan in the nose-bleeds). If you replaced everyone in the Air Canada Centre with this mythical “average” androgynous person you wouldn’t have a hockey game. Remember that oftentimes individuals are very important and the “average” means almost nothing.

Having said this, there are also applications for which flow cytometry is very poorly suited and lie much more appropriately in the domain of microscopy. The most obvious example is with respect to adherent cells and the preservation of morphological features. Flow cytometry requires cells to be in suspension and many preparative methods (i.e. trypsinization) destroy or remove proteins of interest before they can be stained and often makes cells assume a spheroid and sometimes artificial shape. Microscopy further allows for the interrogation of one cell over a period of time, which is something that standard flow cytometers cannot do.