Fluorescence lifetime-resolved imaging microscopy studies: Quantitative image analysis, spectral-flim, and photosynthesis

Development of instrumentation and image analysis methods for the fluorescence lifetime-resolved imaging microscopy (FLIM) were carried out in this thesis. With the new instrument setup and imaging processing algorithms, valuable information has been provided for studying in vivo photosynthesis activities.;Chapter 1 introduces the physics of fluorescence lifetimes and different techniques of FLIM measurements. It also provides practical considerations for successfully acquiring fluorescence lifetime imaging data, especially with in vivo samples. Chapter 2 discusses the image analysis algorithms essential for quantitative analysis of high through-put FLIM data. The advantages of the polar plot, a model-free analysis for fluorescence lifetime data, will be described in detail. The denoising procedure, variance-stabilizing-transform translation-invariant Harr wavelet, and the multi-scale edge detection algorithm, wavelet transform, were applied to improve the precision of lifetime resolution in the images, and to select features at the desired spatial frequency, respectively. K-means cluster analysis was used to analyze a polymer brush microfluid device and the chlorophyll (Chl) a fluorescence transient of Chlamydomonas reinhardtii cells. It will be shown that additional information is provided by the cluster analysis, which would otherwise be hidden in the large quantity of data. Chapter 3 discusses the instrumentation and image analysis methods for the spectrally-resolved FLIM (Spectral-FLIM), which was developed to overcome problems of separating lifetime components in complex environments, especially for fluorescence signals from fluorophores with very low intensity (< 2% of the background). Spectral-FLIM also allows a more detailed and accurate analysis of Forster resonance energy transfer (FRET) measurements. It will be shown in Chapter 3 that a single measurement of Spectral-FLIM can resolve three fluorescence signals (donor undergoing FRET to the acceptor, acceptor excited by FRET, and directly excited acceptor) on the polar plot.;Chapter 4 describes FLIM applications in photosynthesis studies. FLIM data of Chlamydomonas reinhardtii could distinguish the Chl a signals undergoing two different non-photochemical quenching pathways, energy-dependent quenching and state transitions. Spectral-FLIM measurements of avocado leaves (Persea americana Mill.) during the slow phase of Chl a fluorescence transient were also carried out. The data show that the red- and far-red regions of the spectrum have different kinetics and lifetimes, which might suggest that there are two separate binding sites for xanthophyll molecules. Chapter 5 describes experiments to study the lutein-epoxide and violaxanthin cycles operating in parallel in avocado leaves. A good correlation of the fluorescence intensity and Chlorophyll a lifetimes is shown by FLIM measurements, indicating two different quenching states; therefore, the FLIM measurements provide evidence that both cycles are energy-dependent quenching pathways.