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.
Highlights
Fluorescence microscopy is an essential tool for live cell imaging yielding information on macromolecular structure, location, interactions and dynamics
Underpinning tr-fluorescence anisotropy imaging (FAIM), fluorescence lifetime imaging microscopy (FLIM) [2, 3, 28,29,30,31,32,33] maps the fluorescence lifetime in every pixel of an image and is a powerful technique for probing the local environment of a fluorophore as the measured lifetime is largely independent of fluorophore concentration, but can be sensitive to pH [34], refractive index [35,36,37,38], reactive quenching species [39] and viscosity [30, 31, 40,41,42]
We have demonstrated the combination of three powerful fluorescence microscopy techniques - fluorescence recovery after photobleaching (FRAP), FLIM, FAIM - in a single experiment
Summary
Fluorescence microscopy is an essential tool for live cell imaging yielding information on macromolecular structure, location, interactions and dynamics. Underpinning tr-FAIM, fluorescence lifetime imaging microscopy (FLIM) [2, 3, 28,29,30,31,32,33] maps the fluorescence lifetime in every pixel of an image and is a powerful technique for probing the local environment of a fluorophore as the measured lifetime is largely independent of fluorophore concentration, but can be sensitive to pH [34], refractive index [35,36,37,38], reactive quenching species [39] and viscosity [30, 31, 40,41,42] Both fluorescence anisotropy and FRAP have previously been used independently for a study of aggregation states of alpha-synuclein, a protein which plays a role in Parkinson’s disease [43], and an arrangement for dynamic FRAP and rotational diffusion measurements for colloids has been presented [44]. To our knowledge there has not yet been an example of FLIM, FRAP and FAIM used simultaneously in combination for studies of protein dynamics
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