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One consideration when performing multicolor fluorescence studies is the possibility of spectral overlap. Because the fluorophores used in flow cytometry emit photons of multiple energies and wavelengths, a mathematical method called compensation was developed to address the measurement of the photons of one fluorophore in multiple detectors. Due to the nature of flow cytometry measurements, a particle’s emission is measured not in a single detector, but in all the detectors being used in the experiment (Figure 10). For example, FITC emits photons that are green, yellow, and orange, all of which can be detected on a multidetector instrument with the corresponding detectors. In some experiments FITC may be combined with other dyes that emit yellow and orange photons. In those cases the relative contribution of each fluorophore to the signal in a given detector must be determined (Figure 11).
You can avoid the need for compensation by using dyes that don’t have overlapping emission spectra, but this is practically impossible in multicolor flow cytometry.
Figure 10. FITC spillover into other channels. FITC single-stained beads show spillover into PE, PETR, and PE-Cy5 detectors. Gates are set to identify positive and negative populations.
Figure 11. Fluorescence compensation. Emission spectra of two fluorophores commonly used in flow cytometry, FITC and PE are shown. Also shown is a graphical representation of two commonly used filters, 525/50 and 585/40, to detect these fluorophores. Shown in red is the portion of the FITC spectrum that will be detected in the PE detector (585/40) and that must be subtracted from the PE signal using compensation. This process becomes even more complicated when photons from multiple dyes are detected in each PMT.
Which Fluorophores are Useful for Flow Cytometry?
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