Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection

A technology of fluorescence resonance energy and transfer efficiency, applied in fluorescence/phosphorescence, measurement devices, and material analysis through optical means, can solve the problems of difficult measurement and achieve the effect of fast FRET micro-quantitative dynamic imaging

Active Publication Date: 2017-02-22
师大瑞利光电科技(清远)有限公司
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  • Abstract
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Problems solved by technology

Therefore, the actual measurement of this method is very difficult (Seitz A., Terjung S., Zimmermann T. & Pepperkok R. Quantifying the influence of yellow fluorescent protein photoconnersion on acceptor phoyobleaching-based fluorescence resonance energy transfer measurements[J]. J Biom Opt.2012 17(1)011010)

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  • Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection
  • Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection
  • Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection

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Embodiment 1

[0108] 1. Plasmid source

[0109] The donor fluorescent group is the gene-encoded fluorescent protein Cerulean (abbreviated as C), and the acceptor is the gene-encoded fluorescent protein Venus (abbreviated as V). The protein encoded by the reference tandem plasmid C32V is a peptide chain composed of 32 amino acids (TSGLETRDIRSENLYFQGPREFPGGTAGPVAT). The tandem plasmid CTV is Cerulean-TRAF-Venus, wherein TRAF is a receptor-associated factor domain of a long peptide chain tumor necrosis factor composed of 229 amino acids; the tandem plasmid to be tested CVC (Cerulean-5-Venus-5-Cerulean) contains two donors C and one acceptor V, and C and V are connected by 5 amino acids. These plasmids are purchased from the American addgene plasmid bank (Koushik S V, Blank P S , Vogel S S. Anomalous surplus energy transfer observed with multiple FRET acceptors [J]. PloS one, 2009, 4(11): e8031).

[0110] 2. Dual-channel wide-field fluorescence microscope system

[0111] The dual-channel wide...

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Abstract

The invention discloses a method for quickly measuring the fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection. The method disclosed by the invention has the benefits that under the situations that a donor and a receptor are not required to be selectively excited, and the donor fluorescence and the receptor fluorescence are also not required to be selectively collected and detected, fast FRET quantitative imaging detection is realized; the method has low requirements for configuration of an instrument and is low in cost, and exciting light is only required to be switched once in the measuring process, thereby having the advantages that fast real-time dynamic measurement can be performed, and the fast FRET quantitative imaging detection under a millisecond time scale can be realized. Therefore, the method disclosed by the invention has an important application value on FRET quantitative real-time dynamic detection of live cells, thereby certainly greatly promoting the application of an FRET quantitative detection technology to cell biology.

Description

technical field [0001] The invention relates to a method for measuring fluorescence resonance energy transfer efficiency, in particular to a rapid measurement method for fluorescence resonance energy transfer (FRET) efficiency (E) based on dual-channel fluorescence intensity simultaneous detection. Background technique [0002] Fluorescence Resonance Energy Transfer (FRET) refers to the process in which the excited donor fluorophore transfers energy to the adjacent acceptor fluorophore in a non-radiative form when two fluorophores are close enough. FRET needs to meet three conditions: (1) the distance between the donor and receptor is less than 10nm; (2) the orientation of the donor dipole and the acceptor dipole cannot be perpendicular to each other; (3) the emission spectrum of the donor and The excitation spectra of the receptors overlap and the overlapping ratio is greater than 30%. The FRET effect between donor and acceptor is generally characterized by FRET efficiency...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/64G01N21/6486
Inventor 陈同生魏丽春张江
Owner 师大瑞利光电科技(清远)有限公司
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