Abstract
Two-photon absorption (2PA) finds widespread application in biological systems, which frequently exhibit heterogeneous fluorescence decay dynamics corresponding to multiple species or environments. By combining polarized 2PA with time-resolved fluorescence intensity and anisotropy decay measurements, we show how the two-photon transition tensors for the components of a heterogeneous population can be separately determined, allowing structural differences between the two fluorescent states of the redox cofactor NAD(P)H to be identified. The results support the view that the two states correspond to alternate configurations of the nicotinamide ring, rather than folded and extended conformations of the entire molecule.
Highlights
NAD and NADP are the principle biological cofactors involved in cellular redox metabolism.[1]
The hydride-carrying nicotinamide ring is identical in the two molecules, and it is responsible for the spectrally identical intrinsic fluorescence of their reduced forms, NADH and NADPH.[2]
Fluorescence lifetime imaging microscopy (FLIM) is frequently used for this purpose; inside cells, the rate of decay of NAD(P)H fluorescence is dependent upon the enzymes to which the molecules are bound, allowing changes in the metabolic pathways activated in the diseased state to be detected in a label-free manner.[3−8] Maximizing the information content of these measurements requires an increased understanding of how the photophysical quantities reported reflect the biochemical status of the target molecules
Summary
This allows enzyme binding sites to be specific to either cofactor, enabling them to regulate contrasting biochemical pathways. Fluorescence lifetime imaging microscopy (FLIM) is frequently used for this purpose; inside cells, the rate of decay of NAD(P)H fluorescence is dependent upon the enzymes to which the molecules are bound, allowing changes in the metabolic pathways activated in the diseased state to be detected in a label-free manner.[3−8] Maximizing the information content of these measurements requires an increased understanding of how the photophysical quantities reported reflect the biochemical status of the target molecules
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