We have created common human immunophenotyping panels, using 4 fluorophores, which require no compensation. Alternatively you can combine fluorophores that can only be activated by specific individual laser lines, (providing the lasers are spatially separated), but as you increase the number of fluorophores this becomes practically impossible. You can avoid the need for compensation by using fluorophores that do not have overlapping emission spectra. When compensation was not applied, fluorescence spillover can be seen (top panel), which is removed after compensation (bottom panel). Peripheral blood was singly stained with CD4 FITC, CD19 PE, or both CD4 FITC and CD19 PE. Fluorescence compensation corrects for spectral overlap. After compensation we can see that in fact there are no double positive cells, which is to be expected from these mutually exclusive markers.įig.
#COMPENSATION MATRIX FLOWJO SOFTWARE#
The software calculates spillover values and will apply this to the data to obtain correctly compensated data. However when the correct level of compensation is applied using software the true level of staining is revealed. When the sample is stained with both fluorophores, without compensation, a double positive population is observed. Single stained samples reveal the amount of spectral overlap. We can see in Figure 12 how compensation can be applied to a sample stained with antibodies conjugated to FITC and PE. This process becomes even more complicated when photons from multiple dyes are detected in each PMT. 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. Also shown is a graphical representation of two commonly used filters, 525/50 and 585/40, to detect these fluorophores. Emission spectra of two fluorophores commonly used in flow cytometry, FITC and PE are shown. In those cases the relative contribution of each fluorophore to the signal in a given detector must be determined (Figure 11).įig. In some experiments FITC may be combined with other dyes, for example PE, that emit yellow and orange photons. Gates are shown to identify the FITC positive cells and their spillover. FITC single stained lymphocytes show spillover into PE and PE-Cy5 detectors. In some experiments FITC may be combined with other dyes, for example PE, that emit yellow and orange photons.įig. 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 (Figure 10). 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. 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. One consideration when performing multicolor fluorescence studies is the possibility of spectral overlap between fluorophores.
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