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

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. 28-36 (2009

Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

“…We focus on the application of FRAP in this study. However, other imaging techniques such as FLIP or anisotropy imaging of homo-FRET could also be employed in the future using the E-cadherin-GFP mouse to address more fundamental questions regarding E-cadherin regulation at distinct points in the membrane in situ ( Levitt et al., 2015 , Roberti et al., 2011 ). Furthermore, for fine-tuned assessment of E-cadherin via the endogenous E-cadherin promoter, engineering of a knockin E-cadherin-GFP mouse is currently underway as a next-generation mouse.…”

Photon Counting Imaging with an Electron-Bombarded Pixel Image Sensor

“…Single pixel hybrid detectors, which comprise a photocathode in front of an avalanche photodiode (biased below the breakdown voltage), are excellent for photon arrival timing with picosecond resolution [ 21 , 22 ] and are often used in scanning fluorescence microscopy-based FLIM [ 69 , 70 ]. The single photoelectrons liberated by photons at the photocathode are accelerated across a high voltage (8 kV or so) into the avalanche photodiode.…”

Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging

“…We have earlier developed a TR-FAIM approach that enables high-resolution mapping of intracellular diffusivity in relatively translucent, optically homogenous cell cultures 17 18 19 . However, intact animal tissue could be a highly turbid, optically heterogeneous medium which requires a dedicated set of methods for successful implementation of TR-FAIM 20 .…”