What is flow cytometry?
The term "flow cytometry" derives from the measurement (meter) of single cells (cyto) as they flow through a focused light source. In this way various physical and biochemical characteristics of cells may be determined. In many ways a flow cytometer can be envisaged as an automated, rapid, quantitative microscope.
Cells, or indeed any particles, pass rapidly (up to 30,000 cells per second) through a focused light source while contained within a liquid stream. When cells pass through the light source they give out signals, which in turn pass through collection optics and are picked up by detectors. These signals are then amplified and converted into data that can be stored and analysed on a computer. So that different biological or biochemical properties of the cells can be determined at one time, the cells are stained with different fluorescent dyes which bind specifically to the cellular component of interest.
In most modern cytometers today, the light sources used are solid state lasers. Lasers emit light at a specific wavelength, and different types of laser are able to emit light at different wavelengths. As a cell passes through the focused laser beam they will scatter light and emit fluorescence in accordance with the spectral properties of any fluorophores present.
Scattered light is detected in two planes; that scattered in the direction of the laser beam (forward scatter), and that scattered perpendicular to the laser beam (side scatter). Forward scatter gives an indication of a cell's size and side scatter an indication of its shape/texture. The fluorescent light emitted as cells pass through the laser can be quantified and related to the biochemical characteristic of interest.
The vast array of fluorophores available for the measurement of many different biochemical properties of cells makes flow cytometry a very powerful technique.
What is Fluorescent Activated Cell Sorting (FACS)?
In addition to being able to analyse the properties of cells, some flow cytometers are able to sort specific cell populations of interest. These pure populations of cells can then be used in future experiments, cultured or re-stained with other fluorescent dyes and re-analysed.
In most cell sorters, cells are passed in a stream of fluid out through a narrow orifice, at which point they pass through a laser beam and are analysed in the same way as in a standard flow cytometer. A vibration is passed to the sample stream, which causes it to break into droplets at a stable break off point.
If a cell of interest passes through the laser beam it is identified and when it reaches the droplet of the break off point an electric charge (positive or negative) is applied to the stream. As the droplet leaves the stream it passes through deflection plates carrying a high voltage and the droplet will be attracted to one of these plates, depending on the charge it was given.
Uncharged droplets pass through undeflected and deflected droplets are collected in tubes. In this way two different populations of cells can be sorted from one sample. By charging different populations with differing magnituteds of positive and negative charge it is also possible to sort four or six different cell populations from the same sample.
It is also possible to define the number of cells sorted into individual vessles. The most common application for this type of sorting is single cell cloning into 96-well plates.
A more comprehensive guide to the requirements and preparation of samples for cell sorting is described in the 'Techniques' section of this website here.
How is flow cytometric data generated?
During flow cytometric analysis, as a cell passes through the laser beam it produces signals in the form of scattered light or emitted fluorescent light. These light signals (photons) are detected by photomultiplier tubes (PMTs), which convert the signal to a voltage pulse.
The amplitude of this pulse is proportional to the original number of photons. The voltages are then amplified either on a linear or logarithmic scale. Logarithmic amplification increases the resolution of weak signals while at the same time increases the range of data that can be displayed on the same scale. Linear amplifcation on the other hand can indicate subtle changes in a small dynamic range (eg cellular DNA content).
After amplification, voltages are passed to an analogue to digital converter which assigns each signal a specific channel number, proportional to the amount of fluorescence or light scattered. Flow cytometric data can then be scaled in two ways, dependant on whether linear or logarithmic amplification has been used, and the resulting data displayed in a number of different formats.
Presentation of flow cytometric data
The simplest way of displaying flow cytometric data is in the form of a histogram, where fluorescence intensity or the degree of scattered light (x-axis) is plotted against the frequency at which this type of event occurs (y-axis). From such a histogram, the population distribution for the parameter of interest can be ascertained.
To compare the occurrence of two different parameters collected at the same time, bivariate plots are used in which the values for different parameters are plotted on the x- and y-axis. Bivariate plots can be presented in different forms, principally dot, density and contour plots.
In dot plots, a single dot is plotted in relation to the value for x and y for each cell that was analysed. From these plots different populations of cells can be identified, as can rare events, but they give little indication of the population density.
Density plots are similar to dot plots, but the use of different colours enables abundant and less abundant cell populations to be identified.
Contour plots are produced when the points of equal density on dot plots are joined together presenting areas of high density in much the same manner as hills on conventional maps. While these types of plot give an impression for population density, rare events/populations can be lost.