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Proceedings Paper

Confocal FRET and FLIM microscopy to characterize the distribution of transferrin receptors in membranes
Author(s): Horst Wallrabe; Ammasi Periasamy; Ronak Talati; Christine Kim; Margarida Barroso
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Paper 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.

Paper Details

Date Published: 23 February 2006
PDF: 9 pages
Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608905 (23 February 2006); doi: 10.1117/12.661760
Show Author Affiliations
Horst Wallrabe, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Ronak Talati, Albany Medical Ctr. (United States)
Christine Kim, Albany Medical Ctr. (United States)
Margarida Barroso, Albany Medical Ctr. (United States)

Published in SPIE Proceedings Vol. 6089:
Multiphoton Microscopy in the Biomedical Sciences VI
Ammasi Periasamy; Peter T. C. So, Editor(s)

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