Quantitative Measurement System Of Fluorescence Resonance Energy Transfer Based On Emission-spectral Unmixing

Fluorescence Resonance Energy Transfer(FRET)quantitation can allow quantitative analysis of the degree of molecular interaction.With the advantage of high sensitivity,real time measurement and non-destruction,FRET quantitation using emission-spectral unmixing has been regarded as one of the most powerful tool to visualize live-cell protein-protein interactions in real-time.In this thesis,we firstly set up a spectral wide-field microscopic FRET system based on the Independent emission-spectral unmixing method(Iem-spFRET)and the improved Iem-spFRET method(IIem-spFRET).We can combine filters freely according to the experimental requirements in the spectral wide-field microscopic FRET system.This characteristic enlarges the choice scope of the fluorescence probe.Secondly,we built Two-Photon Excitation microscopic FRET system based on Iem-spFRET.We can adjust excitation wavelength from 680 nm to 1080 nm on Two-Photon Excitation microscopic FRET system and this characteristic contributes to lower photobleaching,better penetration and longer detection for samples.Lastly,we implemented Iem-spFRET and IIem-spFRET on the system to quantitatively resolve FRET efficiency(E)and acceptor-to-donor ratio(Rc)of Cerulean,Venus,C32V,CTV,CVC,VCV,VCVV expressed in living HeLa cells.The results show that the spectra of the spectral wide-field microscopic FRET system and Two-Photon Excitation microscopic FRET system can be used for FRET quantification by measuring E and Rc.The results will provide an effective method for real-time optical detection of molecules interaction,which can help to understand the biological mechanism of molecules in living cells.