A software method for color compensation

When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome. Spectral compensation is the process of removing the undesired overlap of signal. Although very successful for two and three fluorochromes, the general practice of adjusting instrument compensation becomes increasingly inadequate and unforgiving as the number of fluorochromes increases. When data are collected uncompensated, software compensation provides the flexibility of setting correct compensation every time for every sample. Software methods do have problems. The linearization assumptions made by the software algorithms may be more or less in error. Binning effects become more of a problem with increasing numbers of compensated parameters. This explanatory unit also contains protocols that illustrate the process of software compensation utilizing matrix algebra that provides for elements of all possible PMT detection combinations. Although details are limited to four colors, the principles described can be applied to any desired number. When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome.