Flow Cytometry Facility

What is Flow Cytometry?

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. The primary laser beam is 488nm and is used to gather the following information:

FSCForward Scatter (FSC) - This is a signal proportionate to the physical size of the particle. As the laser strikes the particle, some light is deflected off the surface but travels within a small angle of the incident beam. The forward scattered light is collected by a photodetector placed in line with the laser, sent to the electronics system, and is normally used as a trigger to begin a data acquisition sequence as well as for its primary purpose of describing cell diameter.
SSC Side Scatter (SSC) - As the particle passes through the laser beam, its internal composition causes the light to bounce around and be refracted in all directions. With an increase in internal organelles, there is an increase in light scattered to all sides. Therefore, the SSC parameter measures the internal complexity of the cell by collecting light scattered perpendicular to the incident beam. As an example, an erythrocyte (red blood cell) has no nucleus and very few organelles so it would have a very small SSC signal. A lymphocyte (white blood cell) has a nucleus and many more organelles, so it generates a higher SSC signal.
IPointFluorescent Signals (FL1, FL2, FL3, etc) - The laser beam excites the fluorophores attached to the particle which causes them to emit light of a different wavelength than that of the laser. These signals are collected in the same direction as the SSC (perpendicular to the incident beam), but pass through a series of long-pass, short-pass, and band-pass filters to allow only certain wavelengths to reach the appropriate detectors. The detectors are photomultiplier tubes (PMTs) which generate electrical signals based on the magnitude of the collected light at its assigned wavelength, as determined by the filter setup. The electrical pulses are digitized and sent to the computer for analysis.

On the MoFlo Astrios, we also have the capability to sort particles. Using the information gathered from the sample using the FSC, SSC, and fluorescent signals, we can identify subsets of particles that may be useful for further study or culturing. In order to do this, we enable the nozzle assembly to vibrate at a very high frequency (approximately 100,000 hertz) which causes the stream coming out of the nozzle to break apart into droplets, each droplet containing one particle. If a particle of interest is detected, voltage is applied to the stream at the exact instant the droplet containing that particle is about to break off from the stream. As soon as the droplet is no longer connected to the stream, the voltage is removed. This results in the droplet containing the particle of interest having an electrical charge while the droplet above it or below it that do not have a particle we want to sort are electrically neutral. (Note: the electrical charge is on the surface of the droplet, not the particle. We do not need to be concerned about the particle itself ever coming into contact with a charge which could damage it.)

cellsortingThis charged droplet then falls between two charge plates which have very high voltage applied to them. Based on the strength of the voltage applied to the droplet, the plates will attract it to either the left or the right at a proportional angle. In this way, the droplets will fall into one of four tubes set up in a vertical array beneath the charge plates. Droplets that are not given an electrical charge are not pulled to one side or the other by the charge plates and fall straight down into a waste tube where it is vacuumed into a waste collection tank for disinfection and disposal.

                                


Flow cytometry is a very robust method of classifying particles. It has a variety of uses and more are being developed as the fluorescent marker industry expands and finds ways to target more specific targets. This is by no means an exhaustive list, but some common applications of flow cytometry are:

  • Cell viability
  • Particle enumeration
  • Cell pigments
  • Immunophenotyping (cell surface antigens, intracellular antigens, and cytokines)
  • Protein expression/modification
  • Transgenic products, particulary GFP or CD markers
  • Apoptosis
  • DNA and RNA content
  • Cell cycle analysis and proliferation
  • Chromosome analysis

Another strength of flow cytometry is that it allows multi-parameter measurements to be taken concurrently and is able to differentiate fluorophores with emission spectra relatively close to each other. One sample can be treated with several different fluorochromes and measured at once to minimize the amount of the sample needed and preserve precious particles. For instance, rather than having to run a million particles for GFP, a million particles for CD45, and a million particles for PI, all three compounds can be found with different emission wavelengths, applied to the same sample, and run once to determine the quantity of each within the sample.

Additionally, flow cytometry allows for high-speed analysis and sorting of a very large volume of particles, into the tens of thousands of events per second. This gives the investigator a statistically significant amount of data to analyze in a relatively short period of time.

Link to Invitrogen’s flow cytometry tutorial (highly recommended viewing)

Last updated on 08-Nov-2016
 
 

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