Flow cytometry is a technique of classifying, enumerating, and sorting microscopic particles such as cells, bacteria, yeasts, picoplankton, chromosomes, and nuclei. A sample is treated with fluorophores that attach to molecules denoting physiological functions or chemical characteristics of the particles, i.e. a protein on the particle’s surface or an intracellular organelle.
This sample is then placed in the flow cytometer. The cytometer is made up of three systems:
- The fluidics system which transports the sample to the interrogation point and hydrodynamically focuses it into a mono-dispersed stream for single event measurements.
- The optics which includes lasers of various wavelength, mirrors, and lenses to focus the laser on the particle stream. This allows the fluorophores to be excited and emit light.
- The electronics system which collects the emitted/scattered light and converts it into proportional electronic signals. These signals are then digitized and processed by the computer for analysis.
The sample is initially placed in the sample chamber where air pressure forces the particles through a probe and tubing to the nozzle assembly. At the same time, sheath fluid (usually PBS) is flowing into the nozzle assembly at pressure slightly less than the sample pressure. This creates laminar flow and a vortex within the assembly which hydrodynamically focuses the sample so that it exits the nozzle vertically separated in a stream of sheath fluid. Essentially, there is a narrow, faster moving, single-file stream of sample within a slower moving stream of sheath fluid. This allows excitation of the sample particles one-by-one.
The laser beams are focused on the stream just below the nozzle. This is called the ‘interrogation point’. As the particle passes through the beam at the interrogation point, it generates multiple optical signals.