Investigation On High-precision Data Acquisition Methods And Algorithms For Fast Fluorescence Lifetime Imaging

Fluorescence lifetime imaging(FLIM)is equivalent to time-resolved fluorescence intensity imaging.Different from traditional fluorescence intensity imaging observing objects'structures with integrated intensity images,FLIM can access microenvironments of molecules including p H,viscosity,temperature,ion,O₂concentrations,protein conformations and interaction by measuring signals'temporal features.FLIM has been broadly used in lifetime science,medical science,physical chemistry,and flow diagnosis.The increasing requirement for observing dynamic phenomena promotes the development of fast FLIM.Various fast FLIM methods proposed previously suffer from the compromise between speeds and lifetime determination precision.Therefore,developing high-precision fast FLIM is essential for determining concerned parameters.This dissertation presented novel approaches for improving the previously proposed fast FLIM methods in terms of precision and speeds.The main contents and innovation points are summarized as follows:1.A rapid lifetime determination algorithm with three time-gates(RLD3)was proposed and a high-precision time-gated FLIM system was established with a wider lifetime determination range than the traditional time-gated FLIM system.With the shortest time-gate's width of 10ns and a coefficient of variance less than 10%,the proposed system has a lifetime determination range of 0.2?40 ns which is wider than the traditional time-gated system(0.4?30 ns)using RLD2.The proposed system can robustly provide FLIM images for slowly moving objects with unknow lifetimes and large lifetime dynamic ranges.The exposure time is long(?0.3 s)as the three images are captured sequentially,which limits the applications for fast-moving objects.Therefore,we developed a high-precision single-shot FLIM method using a framing camera based on the proposed RLD3 algorithm in Part 2.2.A high-precision single-shot FLIM system based on a framing camera was established.Single-shot FLIM experiments for toluene seeded gas mixing jets were conducted.The measured average lifetimes of the whole excited areas(?3×3 mm²)agree well with the results obtained by the streak camera,and they are 7.6 ns(N₂:O₂>7:0.1)and 2.6 ns(N₂:O₂=19:1)with a higher precision than the traditional single-shot FLIM system.With the multiple frames captured by the framing camera in a single-shot and processed with the proposed RLD3 algorithm,the proposed system can provide a lifetime image with a short exposure of 50 ns,a fast lifetime estimation with RLD3(?1 s)and a wider lifetime determination range(0.54?20 ns,compared to the traditional single-shot FLIM system,1.65?20 ns).Due to the limited time-gated images obtained with a framing camera,the proposed system is useful for objects emitting signals following a mono-exponential decay model.However,for signals following multi-exponential and non-exponential decay models,it can only provide(non-amplitude-and non-intensity-weighted)average lifetime images which provide limited information for real applications.If more information is needed,single-shot FLIM methods which can record time-resolved signal profiles should be developed,which is the purpose of Part 3.3.A high-precision single-shot FLIM method based on the space-restricted Compressed Ultrafast Photography method(sr CUP)was proposed with the best performances for complex signals in theory.By using spatially constrained coding,sr CUP overcomes the drawbacks of the compressed ultrafast photography(CUP)method which can only achieve single-shot FLIM for simple signals.For complex signals following a bi-exponential decay model,sr CUP can reconstruct signals with low intensity and strong background noise and can successfully reconstruct lifetime images of the objects with a relative bias<7%and a coefficient of variance<7%for amplitude-weighted average lifetimes and a relative bias<10%and a coefficient of variance<11%for intensity-weighted average lifetimes.In addition,the reconstruction speed of sr CUP is threefold faster than CUP.sr CUP has the potential for observing complex dynamic phenomena in biochemistry and flow diagnosis.4.A histogram classification method and a multiexponential decay visualization method were proposed.The methods can compress the execution times of traditional lifetime determination algorithms(LDA)from several minutes or hours to seconds or sub-seconds for fast estimations and analysis of complex signals.The systems proposed in Parts 1 and 2 use RLD3 processing only three images for a lifetime image with an execution time of?1 s.In contrast,hundreds of time-resolved frames reconstructed with sr CUP in Part 3 or recorded with time-correlated single-photon counting methods can be rapidly processed and analyzed with the proposed methods,which can provide benefits to real-time FLIM in clinical diagnosis and flow diagnosis.