Flow cytometry compensation is a process that corrects for spectral overlap between different fluorescent probes used in a flow cytometry experiment. Spectral overlap occurs when the emission spectrum of one fluorophore overlaps with the excitation or emission spectrum of another fluorophore, leading to signal bleed-through and inaccurate measurement of fluorescence intensities. Compensation involves subtracting the signal from each fluorophore from the signal detected in other channels to correct for this overlap.
Compensation is necessary in flow cytometry experiments where multiple fluorescent probes are used to label different cell populations or biomolecules. These probes emit fluorescence at different wavelengths, which can overlap with other probes or with the autofluorescence of the cells being analyzed. If compensation is not applied, the fluorescence intensities of the different probes will be inaccurate, leading to an incorrect interpretation of the data.
The compensation process is typically performed using control samples that are singly stained with each fluorescent probe or with a combination of two probes. These control samples are used to calculate the spillover coefficient or compensation matrix, which quantifies the amount of fluorescence signal that is detected in each channel due to spectral overlap from the other channels. The compensation matrix can then be applied to the data from the experimental samples to correct for spectral overlap.
Overall, compensation is an essential step in flow cytometry experiments that involve multiple fluorescent probes, as it ensures accurate measurement of fluorescence intensities and improves the quality of the data.
Learn about Sapio Sciences’ flow cytometry data analysis tool here.