Lesson 1: Flow Cytometry Signals (Scatter and Fluorescent)

Before one can begin to design and interpret flow cytometry experiments, a firm grasp of the flow cytometry basics is required. In this lesson we will cover:

  • Cellular Flow
  • “Scatter” signals
  • Fluorescent Signals


Before discussing the details of flow cytometry, let’s first define the terms.

Cytometry: The characterization and measurement of cells and cellular constituents.

Flow: To issue or move in a stream

Flow Cytometry, then, can be defined as the characterization and measurement of cells and cellular constituents as they travel in a stream. In the most basic sense, this is an accurate description. In order to take measurements, a laser beam is focused on the stream as cells travel single-file through the stream.

Hydrodynamic Focusing

In order to make accurate measurements, the cells must be measured one at a time and, therefore, must travel single-file through the stream at the point of laser interrogation. The method of achieving this ordered stream is known as hydrodynamic focusing.

Within the flow cell, a slow-moving sample stream is injected into a faster moving “sheath” stream (generally PBS). Surface tension and laminar flow causes the sample stream to be “wicked off” the injection point into a narrow, faster moving stream within the sheath stream (stream within a stream). Careful control of the velocity of the two streams allow for fine control of the width of the center stream and, therefore, the alignment of the cells within the center stream.

Scattered Light

When the laser beam strikes the stream, the majority of the photons will pass through unobstructed. Some of these photons will diverge slightly, primarily via light diffraction, from their path as they contact the membranes of passing cells. A detector is placed in line with the laser path (on the opposite side of the stream) and this “scattered” light is collected. Because of the nature of its collection, this parameter is referred to as Small Angle Light Scatter (SALS), Forward Angle Lights Scatter (FALS), or, most commonly, Forward Scatter (FSC). Forward scatter is proportional to cell size; the bigger the cell, the more light is scattered, the higher the detected signal.

As cells are translucent, many photons will pass through the cytoplasm. If the photon strikes an organelle (ER, nucleus, etc), the photon will be reflected at a larger angle than those generated by the forward scatter phenomenon. In a typical cytometer, a second detector is placed perpendicular to the laser path to collect light scattered in this manner. This is known as Wide Angle Light Scatter (WALS), Orthogonal Light Scatter (OLS), 90° Lights Scatter, or, commonly, Side Scatter (SSC). Side scatter is proportional to cell complexity; the more organelles/bits inside the cytoplasm, the more light scatter, the higher the detected signal.

Let’s briefly consider the power of this simple technique. If cells are passed through a cytometer with minimal preparation, the cells can already be separated into constituent sub-populations simply by the scattering of the laser light source. The image to the left shows a typical lysed, whole blood preparation. Forward Scatter is plotted on the x-axis with Side Scatter plotted on the y-axis. Each dot represents one cellular event. The scale (0-250) is an arbitrary scale representing increasing intensity of signal. As the image shows, light scatter allows for easy differentiation of small lymphocytes from the larger monocytes and differentiation of the monocytes from the similarly sized but significantly more complex neutrophils. If the cytometer is capable of cell sorting, any of these populations could be separated and collected for further study.


If Flow Cytometry ended with light scatter, it would be a useful technique but certainly would not play its important role in research and clinical science. By adding fluorescent labeling, however, the analysis can be taken beyond morphological characteristics into the arena of functional characteristics. Consider the cell membrane. It is riddled with antigen-specific binding sites. These binding sites relate to specific cell functional roles. For instance, T-cells present CD3 binding sites. B-cells present CD19 binding sites. Therefore, if one took a sample of Anti-CD3 antibodies conjugated with a fluorescent molecule (such as Fitc), one could use the sample to “stain” the T-cells within a sample. As these cells passed through the stream, the laser light would excite the fluorescent tag, or fluorochrome, which would emit photons of light at a higher wavelength (Fitc emits light at ~530nm when excited by a 488nm laser). This light can be collected and used to further categorize the cells. The figure at the left shows a sample which has been stained with Anti-CD3 Fitc, Anti-CD19 PE (~580nm). Notice that the plot shows T-cells (lower right quadrant), B-Cells (upper left), and cells which appear to be neither T- nor B-cells (lower left). Again, if the instrument is capable of cell sorting, these populations could be isolated for further study.

Fluorescence is not limited to antibody conjugation of course. Any source of fluorescence which can be coupled to a functional characteristic is applicable. Intercellular dyes such as Hoechst or Propidium Iodide are used to measure DNA content. Viral transduction efficiency can by measured by the introduction of a fluorescent protein along with the target DNA sequence. The number of parameters than can be measured then is limited only by the availability of distinct fluorescent markers and the specifics of the cytometer hardware (wavelength of installed laser(s), number of detectors, etc).

Wrapping Up If you would like to see the entire process described above, the people at ImmunoTeach have prepared this short movie (quicktime required). In order to asses your understanding of the material thus far, please email the answers to the following questions to the address below. After receiving this I will provide you with access to the next module.

  1. In regards to hydrodynamic focusing, what effect would increasing the speed of the center, sample, stream have on the resolution of signals?
  2. What will be the effect of ethanol fixation of cells prepared for flow cytometry using a green intracellular stain
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