Lifetime and hyperspectral analyses of stem cell nuclei during differentiation

Nicotinamide Adenine Dinucleotide (NADH), is an endogenous regulatory metabolite localised in various cellular compartments including but not limited the nucleus, mitochondria and cytoplasm. The endogenous biological molecule plays a key role in a variety of processes including cellular respiration, transcriptional regulation and differentiation. Through understanding the unique chemical properties and the localisation of NADH in various cellular compartments, can enhance the understanding in determining a cell’s metabolic status and molecular activity. The endogenous properties of NADH are often hard to examine due to their near infrared excitation, transient state within cellular compartments and often uncharacterised binding properties. This leads to difficulty in monitoring real time dynamics in live cells. Described in this thesis are two models, phasor fluorescence lifetime imaging microscopy and spectral phasor analysis which enables the real-time analysis of endogenous NADH inside developing zebrafish embryo somites and living cells. Little is known about the energy metabolite NADHs behaviour in embryology, particularly during somitogenesis, and how the analysis of its spatial location in individual somites may aid in the understanding of the somites metabolic status. Here we describe the use of the phasor FLIM and spectral phasor techniques to spatially map NADH’s lifetime and spectral emissions in developing zebrafish somites and discrete microenvironments in the nucleus of live cells under normal culture conditions and those stimulated to differentiate. Detecting the spectral differences of free and bound NADH in developing tissues and live cells, is currently limited due to the very small differences in emission. The Spectral phasor technique enables not only the examination of small shifts in spectral emissions but also provides the spatial location of spectrally different components in live cells without any prior knowledge of the species being analysed. The phasor representation enables direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells for global analysis. A comparison of undifferentiated stem cells and those stimulated to differentiate demonstrate differing spatial distributions of emission spectral associated with NADH. Undifferentiated stem cells displayed shorter emissions centred in the nucleus, while longer wavelengths were localised around the perinuclear boarder. Stem cells stimulated into the early stages of differentiation displayed a redirection of the shorter emissions to regional clustering predominately in one area close to the nuclear/cytoplasmic broader, while the longer wavelengths were localised throughout the remainder of the nucleus. Here we show the application of the spectral phasor technique to identify discrete wavelength shifts associated with endogenous NADH fluorescence in the nucleus, and observed changes in their spatial distribution in live cells and tissues during the early stages of differentiation. For the first time, this thesis shows the implementation of established statistical methods; the Kruskal-Wallis and Dunn tests, to evaluate the significance of shifts in the ‘g’ and ’s’ coordinates associated with phasor FLIM and spectral phasor analyses. This statistical analysis confirmed the significance of shifts in NADH’s lifetime spectral emissions in myoblasts stimulated to differentiate. Overall this thesis presents the application of two methods used to assess endogenous NADH dynamics. The real-time phasor FLIM and spectral phasor techniques identify the properties of NADH in developing tissues and live cells, that may be applied to fields that assess the metabolic response of living cells and tissues to stimuli, cancer pathology and virology. In addition, this thesis aims to minimise the gaps in current knowledge of NADH’s properties in metabolic activities including tissue development, live cell specialisation and the identification discrete microenvironments within live cells and tissues. The phasor fluorescent lifetime imaging microscopy and spectral phasor techniques give insight into the localisation of specific cellular processes associated with endogenous NADH, in conjunction with further identifying the sequence of key gene events at specific timepoints