The wide-field microscopy requires a high-speed and high sensitivity CCD camera. To allow simultaneous monitoring of spectral emissions at two wavelengths for a donor/acceptor FRET, a dichroic filter is used that reflects the donor excitation wavelength to excite the double-labeled cells and transmit the two emission (donor and acceptor) bands.
For example, for CFP-YFP pair, three cover slips are required to correct the SBT signal from the contaminated FRET image - single labeled donor cells (D), single labeled acceptor cells (A), and double labeled cells (D+A). Cells expressing the CFP- or YFP-fusion protein are initially identified using the appropriate filter sets (Sekar and Periasamy, 2003). An excitation and emission filter wheel is required to acquire the control and the contaminated FRET image. The camera gain, neutral density (ND) filter and image acquisition time are kept constant for all the seven images collected as described in the data process part.
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 FRET data analysis section (Elangovan et al 2003). 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.