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Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine
dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These
coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial
activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this
contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy
imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogenperoxide-
induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon
FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells
with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon
anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental
restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH
exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies
on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in
living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods
that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH
as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to
environmental hazards.
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