High-precision Fluorescence Lifetime Imaging Method And Its Applications

Fluorescence lifetime imaging technique plays an important role in biological environment measurements becauese of its many merits, such as it is sensitive to micro-enviroment, it has high spatial resolution and it is independent to fluorescent concentration and exciting light concentration. Nevertheless, there are some disadvantages of fluorescence lifetime imaging technique based on TCSPC, like low SNR, measurable shortest lifetime is limited to width of the response function, which result in poor measurement accuracy. There are three main aspects including enhancing fluorescence signal, reducing system noise and improving the accuracy of analytical methods in this paper. With these aspects, a high precision fluorescence lifetime imaging method was developed, which has been widely uesd in screening fluorescent mouse sample preparation plan, measuring efficiency of accuracy of FRET and measuring ultrashort fluorescence lifetime. The content is as follows:(1) A simple and practicable method was developed, which is uesd for estimating and optimizing accuracy of fluorescence lifetime imaging system. We estimated spatial resolution of FILM, imaging signal-to-noise ratio and measurement accuracy in quantitative evaluations methods, obtaining conclusions that imaging signal-to-noise ratio is the main factor to affect imaging precision. Thus, two methods-reducing system noise and boosting fluorescence efficiency-was presented to enhance imaging signal-to-noise ratio. The result indicated that detector cooling image signal-to-noise ratio was about4times larger than before and that single photon excitation is much more easier to enhance fluorescence efficiency than two-photon excitation.(2) High precision fluorescence lifetime imaging method was uesd for all mouse sample preparation applications, which is more accurate and faster than fluorescence intensity imaging method. We screened three fixatives and three embedding medium, founding that mouse brain sections of background fluorescence is minimum when we choosed4%paraformaldehyde or GMA. This method has greatly shortened the experimental sample number and experimental period.(3) We optimized our TCSPC-FLIM system for applications in CFP-YFP FRET efficiency measurements. We quantified the FRET efficiency of representative CFP-YFP pairs, and found that the FRET efficiency measurement precision is as high as3%. This study validated that monitoring small FRET efficiency changes is possible with a modified commercial TCSPC-FLIM sytem.(4) We studied the relationship between the shortest measureable lifetime and fluorescence signal intensity and proposed a data analysis method for ultrashort fluorescence lifetime measurement. We compared the classical least squares fitting method (Fitting) with the first moment method (M1) and found that the lifetime measurement precision of the M1method is mainly affected by signal intensity rather than time channel. Further studies revealed that the M1method is better than the Fitting method when the fluorescence lifetime is in the range of70ps to3ns and the signal intensity is less than103photons. Finally, we quantified the ultrashort fluorescence lifetime of CdS nanowires and verified again that the M1method is better than the Fitting method in samples with short fluorescence lifetime. This study provides new technical insights for the quantification of ultrashort fluorescence lifetime or the measurement of small changes in fluorescence lifetime.