Spectral Compensation

When a particle or cell contains several fluorophores (fluorescent molecules) with signals in multiple spectral bands, the identification and analysis becomes considerably more complex because of the likely spectral overlap among the fluoro-phore emissions (Fig. 2.5). For example, a system containing a detector with a band-pass filter designed to collect fluorescence from FITC (fluorescein isothio-cyanate, 525 nm) and another detector designed to collect signals at 550 nm will register photons in both detectors. When a single fluorophore is being collected, this presents no problem, but it is a different story when two or more fluorophores with close emission bands are simultaneously present. It is then necessary to identify which fluorophore was the real emitter of the photons collected on each detector. To achieve this, a process known as spectral compensation must be carried out, whereby a percentage of the signal collected by one detector is subtracted from the signal collected by the other. Of course, the complexity of spectral compensation increases with the number of fluorophores. A special set of circuits must be designed that allows for a varying percentage of each signal to be subtracted from every other detector. While this can be performed perfectly well off-line in software (Bagwell and Adams 1993), if the very goal of the analysis is to sort a certain population of cells, the compensation must be performed in real time between the time the cell passes the excitation beam (interrogation point) and the time it reaches the last point by which a sort or abort decision must be made. Because compensation in FCM is very sophisticated, it requires a large number of controls to establish appropriate compensation settings and

Fig. 2.5 Example of spectral overlap between different fluorophore emissions. Shown in this figure are the excitation lines of several common lasers frequently used in flow cytometers (e.g. violet diode laser (405 nm), argon (488 nm), diode-pumped YAG (532 nm), and helium/neon (HeNe; 633 nm)). Because of the overlap of many fluorochrome emissions, it is necessary to identify and make allowance for the spectral overlap.

Fig. 2.5 Example of spectral overlap between different fluorophore emissions. Shown in this figure are the excitation lines of several common lasers frequently used in flow cytometers (e.g. violet diode laser (405 nm), argon (488 nm), diode-pumped YAG (532 nm), and helium/neon (HeNe; 633 nm)). Because of the overlap of many fluorochrome emissions, it is necessary to identify and make allowance for the spectral overlap.

photomultiplier setup. As fluorescent dyes increase in number and spectral proximity, the need for spectral compensation circuitry becomes more urgent. This is far more involved than anything currently available in image-analysis systems.

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