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Compensation: why and how?

The accuracy and quality of the data is the major factor in using multiple fluorochromes in an experiment. The most critical factor is determining which color should be matched to each antibody in the reagent panel. This is due to a very large range of intrinsic brightness among the fluorochromes commonly used, some antigens being dimly expressed while others brightly expressed, and signals from one reagent optically interfering with signals from another. Whenever more than one marker is expressed on a single cell, the presence of the other fluorescent reagents can contribute significant optical background in proportion to their brightness. This phenomenon is called spillover. The broad band pass and dichroic filters are used to increase the sensitivity in the cytometer in order to separate fluorescence of the emission spectra of dyes from the excitation light source. The emission spectra of fluorescent dyes are broad. For example, fluorescein fluorescence is predominately green and the spectrum contains a range of colors from green to red. It can be seen that some of the light emitted by fluorescein will be pass through the filter used for PE. This is called spectral overlap. Figure 1 shows the emission spectra of some commonly used dyes. While the peak emission is clearly separated for each dye, there is considerable overlap between the dyes. compensation is the process by which fluorescence spillover (overlap) between detectors is mathematically corrected.

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