A multi-component fluorescence lifetime estimation method for fluorescence resonance energy transfer

Abstract

The invention relates to a fluorescence resonance energy transfer multi-component fluorescent lifetime estimation method for measuring the average fluorescent lifetime of all pixel points during fluorescence resonance energy transfer. The method includes the steps of firstly, combining histogram data output by time correlated single photon counting to form a new histogram with few intervals, so part of noise influences are eliminated and the computation amount of an algorithm can be greatly decreased; secondly, conducting autocorrecting computing on interval data of the new histogram, and smoothening an autocorrelation matrix; thirdly, decomposing characteristic values of the autocorrelation matrix to obtain signal sub-spaces; fourthly, conducting matrix division on the signal sub-spaces to form two matrixes, and converting the two matrixes through a rotary matrix; fifthly, estimating the average fluorescent lifetime through computation after obtaining the characteristic values of the rotary matrix. By means of the method, the number of components, the approximate content of components and the approximate fluorescent lifetime information are not needed, and the average fluorescent lifetime information can be directly estimated through two times of matrix characteristic value decomposition and simple conversion.

Description

technical field [0001] The invention is a fluorescence resonance energy transfer multi-component fluorescence lifetime estimation method, and the main function realized is to measure the average fluorescence lifetime of each pixel point during the fluorescence resonance energy transfer. Background technique [0002] Fluorescence resonance energy transfer is an energy transfer phenomenon between two fluorescent molecules that are very close to each other. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is within 10nm, a non-radioactive energy transfer occurs, that is, the FRET phenomenon, so that the fluorescence of the donor The intensity is much lower than when it exists alone (fluorescence quenching), while the fluorescence emitted by the acceptor is greatly enhanced. FRET has become a powerful tool for detecting nanoscale distances and nanos...

AI Technical Summary

Problems solved by technology

Due to the huge amount of calculation of the fitting method, and it is easy to converge to the local extremum, and it is necessary to know the number of fluorescence lifetimes corresponding to each pixel, which is impossible in the experiment, resulting in poor reliability of the estimation results , and use more trial methods, use a variety of models to calculate, and then analyze the results

In recent years, the Prony method, IEM method, CMM method, and Phasor method have been developed with a small amount of calculation, but most of these methods are suitable for single components. For multi-components, it is still necessary to use the fitting method for technical calculations, and Some algorithms use approximate methods, and there are deviations in the estimated results, so the results need to be corrected

Images