Two Photon Fluorescence Lifetime Imaging of Reduced Nicotinamide Adenine Dinucleotide in Brain Research

Current understanding of the structure and functions of the brain is inadequate to explain the complex neurobiological phenomenon associated with the pathophysiology of an organism. Hence, there is a scope for better understanding of the morpho-functional features of the brain. On this note, present-day conventional neuroimaging techniques fail to meet the challenges posed by this complex structured organ to completely unveil the wide array of neurological phenomenon. To make this possible, a novel imaging technique established by combining principles of both two photon excitation and fluorescence lifetime imaging into one system is called two photon fluorescence lifetime imaging microscopy (TP-FLIM). This imaging modality has always been the choice of technique in neurobiology research, as this avenue demands high spatial (~1 µm) and temporal resolution (less than milliseconds). Here, the chapter mainly spotlights on the importance of TP-FLIM of nicotinamide adenine dinucleotide (NADH), a ubiquitous endogenous fluorophore responsible for cellular autofluorescence, and its significance as an indicator of cellular metabolic changes associated with pathophysiological conditions of various disorders. A brief information about the instrumentation of TP-FLIM and fluorescence lifetime measurement methods such as frequency domain and time domain are discussed. NAD+ biogenesis pathway along with its reduced counterpart NADH’s autofluorescence property has been illustrated. Chapter also highlights the application of this technique in cancer diagnosis and in understanding the pathophysiology of neurodegenerative diseases.