My interest is to better understand the technical implications of changing the equipment voltage. Why to do it? When to do it? How to do it? etc.
Hi Peres,
Photomultiplier tubes (PMTs) are optical detectors which detect fluorescence. PMTs read the light which is emitted from the cell crossing the laser. Different fluorochromes will emit light at different wavelengths. PMTs convert light into electrical pulses/signals (i.e. photons are converted into a proportional number of electrons). These signals are digitalized, converted and displayed as "Fluorescence Intensities" in your FACS plots and histograms. Signals can be amplified by applying a voltage to the PMTs, enhancing the electrical current. By changing the voltage you can either enhance or decrease the electrical signal.
Please also check this link: http://medicine.yale.edu/labmed/cellsorter/start/411_66019_Introduction.pdf
Hi Peres,
Photomultiplier tubes (PMTs) are optical detectors which detect fluorescence. PMTs read the light which is emitted from the cell crossing the laser. Different fluorochromes will emit light at different wavelengths. PMTs convert light into electrical pulses/signals (i.e. photons are converted into a proportional number of electrons). These signals are digitalized, converted and displayed as "Fluorescence Intensities" in your FACS plots and histograms. Signals can be amplified by applying a voltage to the PMTs, enhancing the electrical current. By changing the voltage you can either enhance or decrease the electrical signal.
Please also check this link: http://medicine.yale.edu/labmed/cellsorter/start/411_66019_Introduction.pdf
A particular issue comes up when you measure several fluorochromes at the same time. Their emmission spectra usually overlap and within the bandwidth of the filters before the PMTs there might be some fluorescence of the fluorochrome that is close in the emmission spectrum. There are some nice tools online that let you look into this (https://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp)
You have to compensate the signals, which is to define which part of a signal is expected to be derived from another signal. After you have defined these compensation settings, the PMT voltages should not be altered.
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