Study Of Quantitative Fluorescence Resonance Energy Transfer Technology With High Sensitivity

Fluorescence resonance energy transfer（FRET）technique is an important tool to study biological macromolecular interactions in living cells.The independent emission spectra unmixing method（Iem-sp FRET）with high accuracy can be used to investigate the protein interaction in living cells,which makes it outstand among many kinds of quantitative FRET methods.This thesis studied the effect of measurement error on the apparent FRET efficiency,optimized the Iem-sp FRET to improve the measurement sensitivity to study weak interaction between proteins.Firstly,we studied the effect of measurement error on apparent FRET efficiency and receptor-to-donor concentration ratio by introducing random noise in sample data,donor fingerprint,and acceptor fingerprint.The random noise intensity was set from 0.0005 to0.0025,corresponding to 5%-25%of the maximum donor fingerprint intensity.The simulated results show the effect of random noise on apparent FRET efficiency is less than on receptor-to-donor concentration ratio.Random noise with 10%maximum donor fingerprint intensity only leads to 0.33%variation of₁)when the noise is added to both sample and fingerprints.These results indicate that Iem-sp FRET is a robust method.Secondly,we optimized the spectral band and fingerprint normalization method to improve the sensitivity of Iem-sp FRET.The whole spectral band,460nm-620nm is adjusted to three type spectral bands:short spectral band,middle spectral band and long spectral band.The donor and acceptor fingerprint normalization methods include whole spectral area normalization（NW）,partial spectral area normalization（NP）,and maximum value normalization（NM）.The experiment results demonstrate that partial spectral normalization of 460nm-520nm spectral band is the best because the apparent FRET efficiency is almost the same as the theory value and low deviation.Finally,we used the optimized Iem-sp FRET to analyze the fluorescent spectra of nasopharyngeal carcinoma cell CNE1 transfected with A₁-CFP and A_2A-YFP plasmid.The apparent FRET efficiency between A₁-CFP and A_2A-YFP is 0.028,which indicates that there is weak interaction between A₁-CFP and A_2A-YFP.This thesis improve the sensitivity of Iem-sp FRET by optimizing the analysis spectra band and donor and acceptor fingerprint normalization.The optimized Iem-sp FRET is of value to decrease excitation light intensity,photobleaching and to study weak interaction.