This thesis presents the development of fluorescence lifetime imaging microscopy (FLIM) methodologies to advance biological understanding of neurodegeneration and stem cell differentiation. Two FLIM technologies enabling faster FLIM acquisition were developed and tested for high-throughput screening of animal models of neurodegenerative diseases. Each technical implementation was characterised and compared in terms of measurement accuracy, precision, and speed in the screening of C. elegans models of diseases.

Two-photon excitation was combined with FLIM for the study of mitochondrial metabolism in a live-cell model of Alzheimer’s disease. Two-photon excitation enabled the label-free investigation of a key cellular metabolite. The relative abundance of the two binding states of the metabolite were determined with FLIM. This provided a measure of metabolic dysfunction at a single-cell level with sub-cellular resolution. Concurrently, energy levels in the mitochondria were monitored using fluorescent sensors based on Förster resonance energy transfer (FRET). A published approach of delivering exogenous mitochondria to cells was applied to rescue some of the defects seen in the Alzheimer’s disease cell model. The capturing of multiple cellular readouts using quantitative imaging and biochemical assays helped us to uncover the important role proteolytic pathways play in regulating metabolism and protein aggregation.

Finally, a new sensing technique was developed to study chromatin compaction in living cells, based on the fluorescence lifetime of a widely used, far-red, DNA-binding dye. The technique was validated in multiple cell types, tissues, and stem cells. Unlike existing methods (optical or otherwise), our method provided a robust quantitative readout, medium-throughput, and the possibility to study live cells in their native state without genetic modifications. Using this tool, we showed for the first time that pluripotent embryonic stem cells transit through an intermediate decompacted stage before committing to differentiation.

This thesis combines technological developments in FLIM, development of new sensing methods, and multiplexing of known biosensors to gain a better understanding of cellular processes. The entire work has been motivated by biological hypotheses and questions, many of which have been answered by applying our enabling technologies.