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Here, we use fluorescence resonance energy transfer (FRET) imaging techniques to assay and study the organization and dynamics of endosomes in epithelial cells. We use polarized epithelial MDCK cells stably transfected with polymeric IgA-receptor (pIgA-R) to analyze the co-localization, within 10 -100A of pIgA-R-ligand complexes labeled with different fluorophores in the apical endosome. When internalized at 17 degree(s)C for four hours, these complexes co-localize in the apical endosome, which is located underneath the apical plasma membrane. While the transport pathways crossing are thought to be understood, the actual morphology of this endosome has not been completely characterized. Here, we compare the ability of laser scanning confocal and Two-Photon FRET microscopy to image the co-localization of differently labeled (donor: Alexa488, acceptor: Cy3) receptor-ligand complexes in the apical endosome. While the preliminary results are broadly similar, we have found that Two-Photon FRET microscopy possesses significant advantages over laser confocal microscopy FRET. In confocal microscopy FRET, the actual FRET signal in the acceptor channel, following donor excitation, is contaminated by the excitation of the acceptor molecule by the donor wavelength as well as by the cross-talk between donor and acceptor emissions in the acceptor channel. We are in the process of testing an algorithm that will correct this problem. In Two-Photon microscopy, we were able to prevent the excitation of the acceptor molecule by the donor wavelength. Furthermore, we have developed a method to manipulate the Two-Photon FRET data post-acquisition to remove the remaining contaminating signal, i.e. the cross-talk between donor and acceptor emissions in the acceptor channel. Since Two-Photon microscopy avoids out-of-focus bleaching which allows repeated scanning in multiple focal planes, a more detailed picture of intracellular events can be obtained. Therefore, we should be able to use this technology to assay the FRET signal along the vertical axis of polarized epithelial cells, such as MDCK cells. In summary, our results indicate that the Two-Photon FRET microscopy is a powerful tool to assay intracellular protein-protein co-localization/interaction events.
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