Label-free multiphoton microscopy for imaging transient metabolic dynamics in living cells and tissue

Cellular metabolism plays a critical role in human health and homeostasis and is implicated in a large number of pathological conditions. While clinical imaging tools have emerged to probe metabolism at the tissue and organ level, the tools to probe metabolic dynamics at the cellular level have been slow to develop. This thesis represents a step toward realizing one such tool through the use of two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of reduced nicotinamide adenine dinucleotide (NADH). This autofluorescent co-enzyme is involved in both aerobic and anaerobic metabolic processes which can be differentiated utilizing this advanced imaging approach. This thesis first presents a study of cell death dynamics in vivo, with cellular resolution, with a custom-built microscope utilizing a commercial 2P-FLIM detection system. Motivated by the limitations of this study in observing the early dynamics, a high-speed 2P-FLIM instrument is developed and characterized. This developed system is then directly applied to study the rapid, transient metabolic dynamics of cell death, providing new insight into this dynamic metabolic environment. Finally, this tool is combined with fluorescence calcium imaging to study the metabolic dynamics in neuronal activation, revealing a strong cell-specific response to brain activity in dissociated hippocampal cultures. These studies together demonstrate the potential of dynamic metabolic imaging as a tool for both basic scientific research and potential clinical translation. Through further development of these imaging approaches, the complex relationship between cellular metabolism and human health and disease can be further disentangled, providing potential benefits for both future biomedical research and clinical outcomes.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste