![]() The stain index of a fluorochrome defines how 'bright' the emission signal appears to the cytometer. The software then performs a mathematical calculation and the correct amount of compensation is applied to each detector. Each fluorochrome being used is measured in turn and a matrix of spectral overlap is produced. *Image generated using ' BD Fluorescence spectrum viewer '.Ĭompensation measures the amount of spectral overlap of one fluorochrome into the other active detectors on the FACS machine. This is why it is important to have singly labelled controls when setting up your experiment. Compensation removes the non-specific signal produced by a fluorochrome in other detectors. This allows PMT detectors to become highly specific to the particular emission characteristics of different fluorochromes. G lass filters that only allow a defined wavelength range of light are placed in front of the photo-multiplier tube (PMT) detectors. However, in multi-colour analysis it is not possible to avoid spectral overlap so compensation is used to reduce spectral overlap. Spectral overlap can be avoided by choosing fluorochromes that are far apart or excited by different lasers. For example, FITC has its maximum emission at 520nm and PE has its maximum emission at 565nm but the emission spectra of FITC are so wide that some of the FITC signal ends up in the PE detector. Two important things to think about when choosing which fluorochromes to use are the brightness of the fluorochrome and its spectral overlap.Įach fluorochrome emits light in a range of wavelengths and for two fluorochromes there may be a part of the spectrum where they both emit light. Fluorochromes can be conjugated to an antibody made against a marker of interest. ![]() ![]() A fluorescent compound that emits light at a defined wavelength on excitation by a laser.
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