In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Abstract

Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low-and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.T he electron transport chain is the most efficient means of energy production in cells. The electron transport chain produces energy in the form of ATP by transferring electrons to molecular oxygen. The metabolic coenzymes FAD and NADH are the primary electron acceptor and donor, respectively, in oxidative phosphorylation. Neoplastic cells have an increased metabolic demand relative to normal cells because of rapid cell division (1), and neoplastic metabolism is associated with changes in the relative concentrations of NADH and FAD (2-4). Many enzymes bind to NADH and FAD in the metabolic pathway (5), and, as favored metabolic pathways shift with cancer progression, the distribution of NADH and FAD binding sites also change (6). Thus, metabolic imaging of the relative amount of NADH and FAD, and their microenvironment (such as binding sites and/or the presence of local quenchers) can shed light on metabolic changes associated with carcinogenesis.The metabolic coenzymes NADH and FAD are autofluorescent and can be monitored nondestructively and without exogenous labels, using optical techniques. The most common optical method for metabolic imaging is the ''redox ratio,'' which is the ratio of the fluorescence intensity of FAD and NADH (7). This optical redox ratio provides relative changes in the oxidationreduction state in the cell. The redox ratio is sensitive to changes in the cellular metabolic rate and...

A flexible wide‐field FLIM endoscope utilising blue excitation light for label‐free contrast of tissue

“…dents, where cellular autofluorescence lifetimes have been correlated with changes in metabolism associated with cancer, e.g. [12], and in humans, e.g. [13] where studies of skin autofluorescence parameters including lifetime have been correlated with cancer [14,15].…”

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

“…In an ex vivo study, MPT/fluorescence lifetime imaging (FLIM) approach has been shown to be useful for demarcating malignant melanoma and BCC [13]. Metabolic imaging of the relative amounts of reduced NADH and FAD using MPT/FLIM was performed to noninvasively monitor cancer progression in another in vivo study [14]. A MPT/confocal laser scanning microscopy system was used to investigate various dermatological diseases such as actinic keratosis and stucco keratosis [51].…”

“…On the contrary, flavin adenine dinucleotide (FAD) fluoresces in its oxidative state. Hence, FAD and NAD(P)H together can provide ratiometric mapping of cellular redox state [14]. However, the majority of these endogenous skin fluorophores require UV excitation.…”

Diagnostic Modalities for Squamous Cell Carcinoma: An Extensive Review of Literature-Considering Toluidine Blue as a Useful Adjunct

“…Each of our cells contain molecules capable of self-fluorescence, especially when activated (excited) by specific light waves. In humans, these fluorescing products are numerous: tryptophan, porphyrins, collagen cross-links, elastin, NADH (nicotinamide adenine dinucleotide), and flavins (FAD, flavin adenine dinucleotide) [36].…”