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Deprecated: Implicit conversion from float 227.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Biomed+Opt+Express 2015 ; 6 (10): 3842-54 Nephropedia Template TP
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Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells #MMPMID26504635
Levitt JA; Morton PE; Fruhwirth GO; Santis G; Chung PH; Parsons M; Suhling K
Biomed Opt Express 2015[Oct]; 6 (10): 3842-54 PMID26504635show ga
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.