Methods for optimizing PMT voltages on BD digital instruments
In flow cytometry, optimal voltage settings are important for resolution sensitivity (i.e. the ability to resolve dim signals from background noise). While adjusting voltages so that unstained cells appear in the first decade may be adequate for FITC and PE, this method is not optimal for fluorochromes with longer emissions (red to far-red, ~650 nm and longer). This is because the photo-multiplier tube (PMT) detectors are less sensitive to these wavelengths. In addition, unstained cells emit little autofluorescence in this part of the spectrum. Therefore, the variance of dim signals is very high in the red and far-red channels because electronic noise makes a large contribution to these measurements.
The following protocol describes a "Minimal Noise" method for optimizing PMT voltages for your application (i.e. your specific cell type) to ensure that electronic noise makes only a minimal (10-20%) contribution to the measured signals. Once these optimal voltages have been set, you should check that these settings give a good dynamic range for your experimental samples and compensation controls. If needed, PMT voltages can be reduced slightly if some stained samples or compensation controls are off-scale or if they are above the linear range for that detector. Once these adjustments have been made, we recommend running fluorescent beads and recording their MFI for each parameter. By using these bead target values to set the voltages for each PMT in future experiments, you can be confident that day-to-day variations in instrument performance are accounted for to ensure that the resolution sensitivity for each parameter will be consistent from experiment to experiment.
We suggest two "shortcut" alternatives for users who prefer not to manually determine the optimal voltages for their specific application. Further information is available from the links below.