Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Abstract

Fö rster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.Since the discovery of green fluorescent protein (GFP) (1), scientists have been widely employing this gene-encoded fluorescent protein (FP) to investigate the spatiotemporal dynamics of molecules with subcellular resolution. One of the applications of FP engineering is the development of biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) (2). FRET is a nonradiative energy transfer process between two fluorophores located in close proximity to each other, and its efficiency is strongly dependent on the distance between the fluorophores. More precisely, the energy transfer rate (k t ) from a donor to an acceptor fluorophore is calculated in the following Förster's equation;where J is the overlap of donor emission and acceptor excitation spectra, k 2 is an orientation factor of transition moment (a factor determined by the relative orientation of the two fluorophores), n is a refractive index, R is the distance between the donor and acceptor fluorophores, and k f is the donor fluorescence emission speed. Therefore, FRET efficiency is determined by the distance and orientation of the two fluorophores. To measure FRET efficiency, there are roughly two methods; ratiometric analysis and lifetime analysis. Ratiometric analysis utilizes fluorescence intensity of an acceptor (e.g., yellow FP (YFP)) and a donor (e.g., cyan FP (CFP)) upon the donor excitation. Because accept...

Frontiers in intravital multiphoton microscopy of cancer

“…To image intracellular protein/protein interactions and protein activity, numerous molecular biosensors were developed based on the principle of FRET. In FRET, energy transfer occurs between two fluorescent proteins from a donor to an acceptor, and its efficiency is dependent on their local proximity, disappearing at distances greater than 10 nm . Most commonly, FRET is detected by collecting emission signals of donor and acceptor proteins, following donor excitation.…”

“…In FRET, energy transfer occurs between two fluorescent proteins from a donor to an acceptor, and its efficiency is dependent on their local proximity, disappearing at distances greater than 10 nm. 72 Previously, the elevated activity of the small G-protein Rac1 was shown to facilitate the hyperproliferation of cells at the base of intestinal crypts, leading to the initiation of colorectal cancer. 74,75 To test this hypothesis in vivo, a Rac-FRET mouse exhibiting pancreatic ductal adenocarcinoma was developed.…”

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution

“…During FRET, a donor fluorophore is excited and transfers energy to an acceptor fluorophore through a nonradiative process. 75 The energy transfer efficiency is directly related to the intervening distance between the donor and acceptor dyes. Thus, FRET is often referred to as a molecular ruler as it can provide molecular-based distance information between two reporter dyes.…”

Imaging sympathetic neurogenic Ca 2+ signaling in blood vessels

“…The use of optical biosensor mice therefore obviates the need to ‘load’ tissues with a fluorescent dye and provides a phenotypically normal mouse in which artery Ca 2+ signaling (or other) and artery diameter can be readily measured. There are advantages also of biosensor mice, since the problems of transient expression in cultured cells (Hirata and Kiyokawa, 2016), noted for FRET studies, under control of strong (e.g CMV) promoters is avoided; expression in biosensor mice can be stable and non-perturbing.…”