23 February 2006 Confocal FRET and FLIM microscopy to characterize the distribution of transferrin receptors in membranes
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Abstract
Previously, a confocal 'Forster' resonance energy transfer (FRET)-based assay has been used to establish a clustered organization for receptor-ligand complexes containing polymeric IgA-receptor or transferrin-receptor (TFR) during endocytic trafficking in polarized epithelial MDCK cells. Here, the experimental system has been extended to internalizing transferrin (Tfn) labeled with donor fluorophore (Alexa Fluor-488) and/or acceptor fluorophore (Alexa Fluor-555) and applying two-photon fluorescence lifetime imaging microscopy (FLIM)-FRET. The fluorescence lifetime distribution should provide insights, not available with confocal FRET, due to FLIM's ability to reflect the diverse micro-environments of the polarized endocytic pathway. This pilot study confirms that a range of fluorescence lifetime values are detected both in cells containing donor-labeled Tfn (single-label specimens) and cells containing both donor and acceptor-labeled Tfn (double-label specimens) at the level of the basolateral and peri-nuclear common endosomes. Furthermore, significant reduction is detected in the fluorescence lifetime in the presence of donor and acceptor -labeled TFR-Tfn receptor-ligand complexes, when compared with that of donor-labeled, confirming the existence of FRET among these complexes.
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Horst Wallrabe, Horst Wallrabe, Ammasi Periasamy, Ammasi Periasamy, Ronak Talati, Ronak Talati, Christine Kim, Christine Kim, Margarida Barroso, Margarida Barroso, } "Confocal FRET and FLIM microscopy to characterize the distribution of transferrin receptors in membranes", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608905 (23 February 2006); doi: 10.1117/12.661760; https://doi.org/10.1117/12.661760
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