23 February 2006 Confocal FRET and FLIM microscopy to characterize the distribution of transferrin receptors in membranes
Author Affiliations +
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.
© (2006) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
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

Back to Top