Choosing an Acceptable Threshold or Trigger Gate
Choosing an acceptable threshold or trigger parameter and level is important because this determines what the flow cytometer considers to be a valid event and can affect the instrument throughput. The chosen parameter should produce a bright enough optical signal for all particles or cells that are to be analyzed, so they can be easily distinguished from the background noise and debris signals. The better the separation, i.e., the higher the signal-to-noise ratio, the easier it is to set the threshold level to minimize the loss of valid events and the inclusion of noise signals.
Light-scatter parameters (most commonly forward or low-angle scatter) are routinely used to trigger flow cytometer data acquisition. Most eukaryotic cells are large enough and refractive enough to produce a bright light-scatter signal. For example, forward light scatter is a good choice for immunology applications in which cells can be weakly fluorescent (negative) or strongly fluorescent (positive), and for reticulocyte analysis in which the reticulocyte fluorescence ranges from very dim to very bright. In both instances, forward light scatter serves as a reliable means of determining whether a cell has entered the excitation light beam. However, smaller cells, such as platelets and bacteria, and cells or particles that have the same refractive index as the suspending medium would be difficult to see with forward light scatter. For these applications, a fluorescence parameter may be a better choice as a trigger signal. Unfortunately, this may not always be a viable alternative, and compromises need to be made by judiciously setting the threshold level.
Fluorescence parameters can serve either as equally acceptable or as alternative parameters to trigger flow cytometer data acquisition. For some applications, it does not matter whether the system is triggered on light scatter or on fluorescence parameters. For example, the fluorescence intensities encountered in DNA analysis applications are usually bright enough that fluorescence is just as reliable a trigger as a forward light-scatter signal. Bacteria can easily be detected with an appropriate nucleic acid or membrane potential dye, and antibodies coupled to fluorescent dyes can be used to detect a wide range of particles, including bacteria and platelets.
Once the parameter for triggering flow cy-tometer data acquisition has been selected, the level of the trigger needs to be set. Ideally there would be a sufficient separation between the cells of interest and the background noise so the threshold level could have a wide range of acceptable values. However, in some instances, separation between noise and signals is minimal, so the threshold is more difficult to set. If it is set too high, some of the particles or cells of interest are missed. If it is set too low, then the data acquisition system spends more time analyzing noise and, as a result, some of the particles or cells of interest are missed. The investigator needs to set the threshold for the highest sample throughput of the particles of interest which minimizes missing of the particles or cells.
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