In Multiphoton FRET (MP-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.
If the donor and acceptor excitation wavelength is not known and here is the procedure one would follow to identify the excitation wavelength for the selected FRET pair: For MP-FRET, the ti:sapphire laser is tuned to detect the maximum and minimum signal for the donor and acceptor proteins expressed individually. The wavelength corresponding to maximum donor signal and minimum acceptor signal will be used to collect the FRET signal from the doubly expressed cells. For example, in the case of cells expressing C/EBPD244 protein tagged with CFP (donor) and YFP (acceptor), the laser wavelength is tuned from 700-1000 nm. The maximum CFP signal and minimum YGFP signal are seen at 820 nm. Maximum YFP signal and minimum CFP signal are seen at 920 nm. Hence, an excitation wavelength of 820 nm is used to acquire the FRET images from doubly expressed (CFP-YFP-C/EBPD244) cells. This method of selecting donor and acceptor excitation wavelengths can be used for any possible MP-FRET fluorophore pairs. Since the selected donor excitation wavelength can also excite (about <10%) the acceptor fluorophore molecule present, the technique requires correction to remove unwanted fluorescence signal in the FRET image (Elangovan et al., 2003).
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.