Cytometry 101

aspect ratio pixel

Welcome to FlowJo University!  Before we begin the discussion on how to use FlowJo to analyze single cell data, lets first discuss where that data comes from.  Single cell data can be collected using flow cytometry: the measurement of cells in a flowing medium.  The tool used to make these measurements is called a cytometer.  Cytometers, with a few examples shown here, come in all shapes and sizes but they share some underlying principles. Cytometers are generally comprised of fluidics, optics, and electronics systems. The fluidics system accepts the cell sample (colored red dots in this illustration) and transports it to the interrogation point, while forcing the cells into a single file stream though either hydrodynamic or acoustic focusing.  

The optics system is then engaged.  At the interrogation point, where the blue laser hits the red cells in the graphic, the cells pass through a laser beam or beams that excite florescent probes bound to selected cellular targets, emitting light of a different color for each unique probe.  That light is filtered into individual colors and the quantity of emitted photons is measured using photomultiplier tubes, or PMTs. The quantitative aspect of this is important; cytometry measures relative ammounts allowing for measurement of the intensity of expression instead of simply yes/no or on/off. 

Finally, the electronics system is used to convert excited photons to voltage measurements to a singular number representing intensity of expression.

Lets take a closer look at how the numbers we work with are produced...

One topic glossed over so far is how multiple phenotypes are identified.  Monoclonal antibodies tagged with the aforementioned florescent probes are used to bind specifically to the selected antigen that will reveal the phenotype under study.  Cytometry allows for the measure of up to approximately 50 different cellular aspects depending on the system. The measurement of these multiple aspects on many cells are then stored as a flow cytometric standard file, abbreviated as FCS.  FlowJo will accept FCS files from any cytometer.

That concludes the introduction to cytometry.