Quantitative Ratiometric Imaging of FRET-Biosensors in Living Cells

Abstract

Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.

Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences

“…Since there are many biosensors based on two compatible FRET pairs of FPs (Miyawaki et al 1997;Itoh et al 2002;Kurokawa et al 2005;Mitra et al 2005;Kawase et al 2006;Pertz et al 2006;Cai et al 2008;Ouyang et al 2010;Zawistowski et al 2013;Hanna et al 2014;Moshfegh et al 2014), their expression profiles in stably integrated cell systems must be carefully assessed by immunoblotting and FACS analysis. Indeed, in our previous works, several approaches were taken to minimize potential problems by carefully (1) optimizing the biosensor induction condition and duration, (2) analyzing the expression patterns of the biosensor on Western blots and FACS sorting for limited populations of cells that express both CFP and YFP, and (3) choosing, critically, cells expressing both FPs at near-expected intensities during single-cell microscopy imaging (Hodgson et al 2010;Spiering et al 2013). Especially in light of the recombinant deletion frequency being a function of the insert size between the two homologous components such as the FPs ) example construct as shown in F was transfected into MEF3T3 fibroblasts under a tet-OFF-inducible system and was selected for stable integration using an antibiotic selection.…”

“…Cells were imaged using a custom, optimized multichannel epifluorescence microscope (Spiering and Hodgson 2012) under a 60× 1.45 NA DIC oil immersion objective lens. The FRET and mCer channels were acquired simultaneously using two Coolsnap ES2 cameras (Photometrics) mounted on the side of the Olympus IX81 microscope via an optimized beam splitter, allowing for simultaneous image acquisition to eliminate motion artifacts, as previously described (Spiering and Hodgson 2012;Spiering et al 2013). The third camera, mounted on the bottom port of the same microscope, acquired the matching DIC image set.…”

FRET Imaging in Three-dimensional Hydrogels

“…This is of great concern for probes that localize to cellular structures, such as the plasma membrane EPAC1 probe PM-ICUE. See Spiering et al 2013 for suggestions to correct for cell thickness effects on ratio calculations 24 . Analysis of the distribution of Aem can be used to detect if probe accumulates in regions of the cell and imaging plane adjusted.…”

“…"Single-chain" ratiometric probes utilize a fluorophore pair linked on a single molecule. "Dual-chain" probes utilize fluorophore pairs on separate molecules, but their expression can be coupled under the same expression cassette 24 . Unlike studies with single fluorophore expression (e.g., fluorescent fusion proteins), studies with ratiometric FRET probes do not require dual transfection to control for transfection efficiency or cell viability.…”

“…If the fluorophores of "single-chain" probes are of similar brightness (function of extinction coefficient and quantum yield), the effects of non-linear detector sensitivity are small and only correction for the background fluorescence and the coupled quantum yield/detector sensitivity is needed 23 . Thus "singlechain" ratiometric FRET probes are most easily implemented in widefield microscopy 24 . This paper describes a straightforward technique to perform FRET imaging of live cells in 3D hydrogel cultures using a conventional widefield epifluorescent microscope.…”