Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells.
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.
Department of Physics, King's College London, Strand, London WC2R 2LS, UK.
James A
JA
Levitt
Division of Asthma, Allergy, and Lung Biology, Guys Campus, King's College London, London, UK ; Randall Division of Cell and Molecular Biophysics, Guys Campus, King's College London, London, SE1 1UL, UK.
Penny E
PE
Morton
Department of Imaging Chemsitry and Biology, Division of Imaging Sciences and Biomedical Engineering, St. Thomas Hospital, King's College London, SE1 7EH, UK.
Gilbert O
GO
Fruhwirth
Division of Asthma, Allergy, and Lung Biology, Guys Campus, King's College London, London, UK.
George
G
Santis
Department of Physics, King's College London, Strand, London WC2R 2LS, UK.
Pei-Hua
PH
Chung
Randall Division of Cell and Molecular Biophysics, Guys Campus, King's College London, London, SE1 1UL, UK.
Maddy
M
Parsons
Department of Physics, King's College London, Strand, London WC2R 2LS, UK.
Klaus
K
Suhling
(170.2520) Fluorescence microscopy; (170.6920) Time-resolved imaging; (180.0180) Microscopy; (260.2160) Energy transfer; (260.5430) Polarization; (300.0300) Spectroscopy