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Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

Research output: Contribution to journalArticle

Original languageEnglish
Article numberA025
Pages (from-to)3842-3854
Number of pages13
JournalBiomedical Optics Express
Volume6
Issue number10
DOIs
Publication statusPublished - 8 Sep 2015

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  • boe_6_10_3842

    boe_6_10_3842.pdf, 2.07 MB, application/pdf

    15/12/2015

    Final published version

King's Authors

Abstract

We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells.

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