Analog threshold gating is typically done on only one optical parameter. A single analog comparator is used to detect when a pulse amplitude exceeds the threshold value. The threshold parameter can be either a light-scatter signal or a fluorescence signal. The choice of parameter depends on which parameter gives the best signal-to-noise ratio, i.e., the best separation between noise and signal. Forward light scatter is the most common signal used as it is typically a reliable indicator of when a cell or particle is intersecting the excitation beam. If the cells or particles are large enough and have a refractive index different enough from that of the suspending medium, then it is likely that they will have a strong enough forward light-scatter signal to be used as a trigger.

Forward light scatter is particularly useful for applications in which the sample consists of particles ranging from nonfluorescent to brightly fluorescent. For those types of samples, the amplitude of the fluorescence signal may not always be large enough to trigger the threshold gate; thus, some cells or particles may not be counted. Using forward light scatter as a trigger ensures that all the particles or cells have their fluorescence quantified correctly.

A fluorescence parameter is used as a trigger when all the particles have sufficient fluorescence intensity to trigger a threshold gate, e.g., DNA analysis. Also, if the amplitude of the forward light-scatter signal is very small, as when the particles are very small or have an index of refraction very nearly that of the suspending fluid, then a fluorescence parameter is a viable alternative trigger if the cells or particles can be stained brightly enough with a fluorescent dye. A good example of this scenario is bacteria stained with a fluorescent dye. Bacteria are typically very difficult to detect with forward light scatter in a nonepi-illumi-nated flow cytometer; thus, staining the bacteria with a fluorescent dye makes them bright enough to reliably trigger a fluorescence-signal threshold gate. The fluorescent dye can be a nucleic acid dye, a membrane potential dye, or an antibody-coupled dye, to name a few examples. The key is to pick a bright dye that does not bind significantly to debris or other contaminating particles.

Another interesting way to use a fluorescence parameter as a trigger is for processing or sorting rare cells if the rare cells are the only brightly stained cells and a count of the other cells is not required. Gating on only the brightly stained rare cells means that flow cytometer data acquisition would trigger just on the few bright cells of interest and ignore the rest. Few, if any, bright rare cells would be missed, making this a simple way to analyze and/or sort just the rare cell population (McCoy et al., 1991). Note, however, that the resulting sorted samples, although enriched, would have a low purity. Because the threshold gate is set high enough that only the brightly stained rare cells are analyzed, other contaminating cells and debris are ignored by the flow cytometer sorting-decision logic.

In designing an application to be run on a flow cytometer, it is important to have a reliable means of triggering the flow cytometer so as to analyze all the desired cells or particles and minimize false triggering of the system by noise signals. Though forward light scatter is the most common signal used to trigger the threshold gate, fluorescence parameters can be used as well and are required in some applications. The key is to identify an optical signal with a high signal-to-noise ratio that is possessed by all the cells to be included in the analysis.

0 0

Post a comment