Fluorescence Resonance Energy Transfer (FRET) technology based on different optical platforms

: Fluorescence Resonance Energy Transfer (FRET) is an effective method for qualitative and quantitative detection of interactions between biological large molecules. According to the fluorescence
scope
configuration and different application focus, can be in the solution, cell suspension, multicellular, single cell, cell membrane, cell device and other different levels of interaction distance between biological macromolecules, dynamic properties and so on. In this paper, FRET methods based on conventional wide-field fluorescence microscopes, full-reflection fluorescence microscopes, continuous laser concentration microscopes, pulsed multiphoon excitation microscopes and other microscopes are introduced and evaluated respectively. Reference.10 is relevant for laboratory applications and configurationIntroduction: Fluorescence Resonance Energy Transfer, W-FRET, TIRF-FRET, Confocal-FRET, MP-FRET1 Introduction With the deepening of life science research, optical microscopes enable us to understand cellular structure and related functions. However
molecular biology
studies have shown that molecular events, such as signaling and gene translation, require the assembly of
proteins
into special large molecular complexes. The study of the mechanisms by which various life phenomena occur, especially the interaction of proteins within cells, has become particularly important. Traditional biophysical or
biochemistry
methods, such as affinity
chromatography
or immunosuppression reaction and the recent
yeast
dual hybridization, phosphatization
antibody
, immunofluorescence, radioactive markers, etc., all require broken cells or damage to cells, and cannot be carried out in real time under living cell physiological conditions of protein-protein interaction dynamic research.intensity-based imaging techniques such as the FRET method (wide field, cofocus, dual photons) make it easier to study these interactions within living cells (Periasamy, 2001). The new imaging technology, combined with the development of new gene-coded fluorophore markers and sensors, as well as computer image acquisition and analysis software, enhances the capabilities of many complex studies such as protein function and processing, gene expression and second messenger transmission, and in-cell signaling. (Roessel and Brand, 2002).FRET is the process of transferring the excitation state energy of the supply to the subject excitation state through the interaction of the electrodes between molecules, which is a non-radiation leap, and when FRET occurs, the distance between molecules is less than 10nm. In the event of FRET, the supply path signal will be quenched and the subject path signal will be activated or enhanced (Herman, 1998). FRET microscopy relies heavily on the ability to quickly and efficiently capture short, weak fluorescent signals from the interactions between calcing molecules. Since the energy transfer occurs at 1-10nm, a FRET signal represents a special position in a microscope image. This is equivalent to providing an additional magnification that allows FRET to distinguish the average distance of the supply-receptor at a molecular scale beyond the resolution limits of the microscope and to show the interaction of the receptor-to-feed. 2 FRET microscopic technology based on different optical platforms FRET feeder-subject pairing a widely used feed-subject fluorophore in FRET studies comes from the spontaneous fluorescent protein GFPs. To select GFPs as a working FRET pair, careful consideration should be given to its
spectral
characteristics: pairing the selected supply subjects, their excitation spectra should be sufficiently separated, the feed emission spectrum overlaps the subject excitation spectra (>30%) to obtain sufficient energy transfer, and the feed member's emission spectrum is reasonably separated to independently measure the fluorescence of each fluorescent group. , depending on the biological application, there are some combinations of FRET pairs. CommonLYT pairs of fluorophores are: CFP-YFP; CFP-dsRED; BFP-GFP; GFP-dsRED; YFP-dsRED; Cy3-Cy5; Alexa 488-Alexa 555; Alexa 488-Cymed3; FITC-Rhodamine ;Y-TRITC; Y-Cy.FreT method configurationFRET method can be based on different optical platform configuration, the application is very wide. The general FRET system consists of the following configurations:excitation light sources: stable mercury lamps, xenon lamps, mercury xenon composite arc light sources, and laser light sources at different wavelengths from ultraviolet to infrared bands.neutral density filter: used to control the intensity of the excitation light.filter group: configure the appropriate filter set for the selected fluorophore combination, including excitation, emission, two-color dlight, etc. The filter combination needs to be carefully selected to reduce the spectral string color and increase the signal noise ratio of the FRET signal.detection equipment: high sensitivity PMT or cold CCD.the optical platform configuration used, FRET microscopy can be divided into two categories: single photon excitation and multi photon excitation. Single photon excitation classes include Wide-Field-based W-FRET, Full Internal Reflection TIRF-FRET, and Confocal C-FRET. Multi-photon excitation includes dual photons and multi-photons (MP-FRET).