Flow cytometry is a method of single-cell analysis that includes the characterization of a cell's physical properties. In a flow cytometer, a cell population is suspended in a clear saline solution. The suspension is funneled through a nozzle that forges a single-cell stream. The population then flows past a set of laser light sources one cell at a time. While being interrogated by the lasers, the cell scatters light (1). Figure 1 provides an overview of a flow cytometer schematic.
Figure 1. Flow cytometer optics schematic (2).
The ratio between cell size and laser wavelength alters scatter behavior. When cell size is smaller than the excitation of the interrogating laser, the scatter behavior is inconsistent and low-intensity. Accordingly, the laser usually emits light at a wavelength shorter than interrogated particles. A wavelength of 488 or 405 nm is common (1).
The light scatter is measured by two optical detectors. One detector measures scatter along the path of the laser (1). This parameter is referred to as forward scatter (FSC). The other detector measures scatter at a ninety degree angle relative to the laser (1). This parameter is called side scatter (SSC). When measured in conjunction, these two measurements allow for some degree of cellular differentiation within a heterogeneous population.
The measurement of forward scatter allows for the discrimination of cells by size. FSC intensity is proportional to the diameter of the cell, and is primarily due to light diffraction around the cell. Forward scatter is detected by a photodiode, which converts the light into an electrical signal. The intensity of the produced voltage is proportional to the diameter of the interrogated cell (1).
FSC is helpful for distinguishing between cells of the immune system. Monocytes and lymphocytes are two classes of white blood cells. In general, monocytes are larger than lymphocytes and exhibit forward scatter of a higher intensity.
Figure 2. Discrimination of lymphocytes and monocytes in FlowJo™ based on scatter parameters. Note: This example is for illustration purposes only, the populations illustrated are either not actual monocyte/lymphocyte cells, or the patient is exhibiting acute monocytosis.
Side scatter measurement provides information about the internal complexity (i.e. granularity) of a cell. The interface between the laser and intracellular structures causes the light to refract or reflect. Cellular components that increase side scatter include granules and the nucleus (1).
Relative to forward scatter, light signals from side scatter are weak. A photomultiplier tube (PMT) is used to measure side scatter because it is a more sensitive optical detector (1).
Side scatter is helpful for identification of cells with varying complexity. For example, monocytes and granulocytes. Granulocytes are characterized by the high volume of cytoplasmic granules. The light reflection off of the granules increases the intensity of the SSC measurement and allows for discernment between granulocytes and monocytes (1).
Figure 3. Discrimination of granulocytes and monocytes in FlowJo based on scatter parameters.
For every signal (i.e. electromagnetic pulse) which passes into a detector (PMT or photodiode) there are three characteristics which can be recorded: Height, width and area. While the pulse area is most commonly reported, there are distinct advantages and disadvantages to each. Within scatter parameters, the pulse height versus pulse width plots are used to isolate single cells passing through the cytometer, and thereby remove any non-single cells (doublets, clumps or debris).
Figure 4. Discriminating single cells from scatter height versus scatter width parameters in FlowJo™.
A flow cytometer measures FSC and SSC concurrently, and the two parameters combined provide a fairly good foundation to begin analysis of a cell population. There are further methods of cell quantification, but there is a great deal of information that can be obtained from scatter data.
Shapiro, Howard. Practical Flow Cytometry. New York, Alan R. Liss, 1985.
Schematic overview of a typical flow cytometer setup. 2016, SelectScience.
Some Additional Resources to assist in understanding FSC vs SSC