INTENSITY-BASED ENERGY-TRANSFER MEASUREMENTS IN DIGITAL IMAGING MICROSCOPY

Investigation of protein-protein associations is important in understa nding structure and function relationships in living cells. Using Fors ter-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membr ane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy t ransfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for bac kground correction of images. In addition to the reference autofluores cence images (UV background) we recorded three fluorescent images (don or, acceptor and energy transfer signal) of donor-acceptor double labe led samples, and corrected for spectral spillage of the directly excit ed donor and acceptor fluorescence into the energy transfer image. Aft er careful image registration we were able to calculate the energy tra nsfer efficiency on a pixel-by-pixel basis. In this paper, we also pre sent a critical comparison between results obtained with this method a nd other approaches (photobleaching and flow cytometric energy transfe r measurements).