Imaging molecular interactions in living cells by FRET microscopy

Abstract

Förster resonance energy transfer (FRET) is applied extensively in all fields of biological research and technology, generally as a 'nanoruler' with a dynamic range corresponding to the intramolecular and intermolecular distances characterizing the molecular structures that regulate cellular function. The complex underlying network of interactions reflects elementary reactions operating under strict spatio-temporal control: binding, conformational transition, covalent modification and transport. FRET imaging provides information about all these molecular processes with high specificity and sensitivity via probes expressed by or introduced from the external medium into the cell, tissue or organism. Current approaches and developments in the field are discussed with emphasis on formalism, probes and technical implementation.

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

“…Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules (Depry and Zhang, 2010;Hodgson et al, 2008;Jares-Erijman and Jovin, 2006;Kiyokawa et al, 2010). Through the efforts of many research groups, FRET biosensors have been created to visualize the changing concentrations of ions, sugars, and phospholipids, and to monitor the activities of GTPases, protein kinases and proteases.…”

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

“…IN biological systems, fluorescence resonance energy transfer (FRET) is a widely used method for studying molecular interactions and processes (1). FRET is a nonradiative transfer of energy from an excited fluorophore, the donor, to another fluorophore, the acceptor, that are at a distance of 1-10 nm (2).…”

“…The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,(4)(5)(6)(7)(8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations.…”

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

“…Of these, Förster resonant energy transfer (FRET) is probably the most widely used approach to map protein binding and the readouts of genetically expressed biosensors that change their molecular conformation upon binding their analyte. Many techniques have been implemented to read out and quantify FRET [15,16] with the most widely used being spectral ratiometric imaging, where FRET is read out through the change in acceptor to donor intensity ratio, and fluorescence lifetime imaging (FLIM), where the increased de-excitation rate resulting from FRET results in a decrease in the donor fluorescence lifetime. While the former approach typically requires fewer detected photons per pixel, quantitative spectrally resolved FRET measurements require calibration of the spectral response of the system (including sample and instrument) and an independent measurement of the actual FRET efficiency is also required if the fractions of the FRETing donor and acceptor populations are needed [17].…”