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Flow Cytometry at San Jose State University

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Flow Cytometry

A flow cytometer is an instrument that analyzes cells as they flow in a liquid stream through a beam of light. Flow cytometry techniques record characteristics for each cell flowing in the stream and are capable of analyzing thousands of cells in a matter of seconds. Characteristics may include size, shape, the ability to reflect or scatter light, and fluorescence. Fluorescence emission can result from autofluorescence from endogenous sources (such as storage granules and chloroplasts), or from fluorescent dyes used experimentally to “tag” a cell or cellular structure. Fluorescent tags can be associated with cells nonspecifically (e.g. labeling proteins or nucleic acids), or specifically (e.g. using a specific antibody or nucleic acid probe).

During collection of flow cytometry data (called ‘acquisition’), cells move in a single file and cross the path of a laser beam. When the light hits each cell, some of the light scatters. If the cell is tagged with a fluorescent dye, the light hitting the dye excites the dye and fluorescent light is emitted at a specific wavelength. Several fluorescent dyes, each used to tag  a different cellular component, can be detected simultaneously. All of the light data generated from the laser beam hitting the cell is recorded and transferred to a computer for analysis.

There are several basic components of a flow cytometer, as follows:

·           A laser light source (488nm)

·           A stable optical bench to focus and direct the light (keeps the light source and the particles being observed in alignment)

·           Fluid lines and controls to direct and regulate the flow of liquid stream containing cells through the light beam

·           Electronic network for measuring and recording the intensity of light signals

Figure 5: Simplified layout of a typical analytical flow cytometer.

Typically, in a flow cytometer, cells flowing in a liquid stream are hit by a light beam generated by an argon laser. When the light leaves the laser, it is focused into a beam  about 50µm in diameter and approaches the liquid stream which has a 50-150µm diameter and flows perpendicularly to the light beam. The light beam crosses the liquid stream at the interrogation point (Fig. 5). Lenses surrounding the interrogation point collect the light emerging after it passes through the stream. This emerging light is focused onto photodetectors (photodiodes or photomultiplier tubes) that convert the light into an electronic signal. A typical configuration for photodetectors is three to five photomultiplier tubes at right angles to the beam. The photodetectors are at the side and not in the direct path of the beam. They receive light either deflected or scattered by the cells in the stream.

One of these photodetectors has a blue filter. The light illuminating the stream is a blue light. If any light is bounced or scattered from the particles in the stream it will be captured as “side scatter” or SSC light (the light of the same color as the illuminating beam). This light is a measure of the texture of the particles flowing across the light beam, sometimes called the “granularity signal”. For example, when blood cells are analyzed, the granulocytes with irregular nuclei have larger SSC than erythrocytes (anucleate red blood cells). Other photodetectors are situated at right angles but have green, orange, and red filters that detect emission signals from fluorescent dyes used to label cells. Since each of these 'fluorescence' photodetectors only captures the emission wavelength of one dye, each emission signal is captured independently.

Another photodetector captures signals directly in the path of the beam. However, this forward angle photodetector has a block that prevents the beam of light from impinging directly on the photodetector. Therefore, any light that does hit this forward photodetector arrives by being bent as it passes through a particle. An angle of 0.5° generates a signal. This signal is referred to as the Forward Scatter (FSC) or Forward Angle Light Scatter (FALS), which is light of the same color as the illuminating beam. This signal is related to the volume and the refractive index of the particle. Dead cells bend light less than a viable cell. Thus, they give a dimmer FSC compared to living cells due to leaky outer membranes which, in turn, causes the refractive index to be similar to its surroundings. In this manner, forward (FSC) and side scatter (SSC) are used to exclude debris, dead cells, or cell aggregates in an experimental analysis or, in the clinical laboratory, are used with blood specimens to distinguish lymphocytes, monocytes, and granulocytes.

The fluid flow system is an important component since it allows the cells or particles to move in a single file as they encounter the light beam. The movement of the particles is controlled by the flow of the sheath fluid, a buffer that may be distilled water or phosphate-buffered saline. A flow rate of 1000 particles per second is typical. Flow cytometers generate large amounts of data as they rapidly and simultaneously determine the relative value of each parameter for each cell within large cellpopulations These data must be analyzed with the aid of a computer to extract meaningful information. Flow cytometry data are often represented in a dot plot (Fig 6). The dot plot quantitates the relative intensity of various parameters for a population of cells.

Figure 6: Dot plot of cells stained for CD3 and CD8 cell surface proteins. Each dot represents a single cell. As the intensity increases, the cell is positive for the indicated marker. Cells in the upper right quadrant are positive for both the CD3 and CD8 proteins.