In Confocal FRET imaging, we select the appropriate filters and high sensitivity photomultiplier tubes (PMTs) to acquire donor and acceptor images. It is important to note that appropriate average power should be used to reduce photobleaching.
The background subtraction of the image is important to remove the autofluorescence, detector and optical noise. The SBT correction should be implemented as discussed in the data process part. Seven images are required. In brief, (1) single labeled donor cells should be excited with donor molecule excitation wavelength and D- and A- channel images are acquired. (2) Single labeled acceptor molecule should be excited with donor and acceptor wavelength and the A- channel images are acquired. (3) Double labeled (D+A) cell should be excited with donor excitation wavelength and the D- and A- channel images are acquired. Acceptor excitation wavelength will be used to excite the D+A labeled cells and collect the A-channel image. These seven images are used toprocess to obtain the processed or precision FRET (PFRET) image.
The laser power for excitation for donor and the acceptor may be different. But once you adjust the donor laser power (say 10%) and that should be used whenever you use the donor excitation wavelength. The same way the acceptor excitation wavelength, if you use, say 5% or 10% for the acceptor excitation wavelength then, the same acceptor power (5% or 10%) should be used whenever you use the acceptor excitation wavelength.
The same theory for PMT gain adjust for donor and acceptor emission. It is important not to saturate the pixel intensity.