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.

https://www.pnas.org/content/pnas/104/49/19494.full.pdf

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

Purpose. To characterize the intrinsic fluorescence (autofluorescence) of the human ocularfundus with regard to its excitation and emission spectra, age relationship, retinal location,and topography, and to identify the dominant fluorophore among the fundus layers.Methods. Using a novel fundus spectrophotometer, fluorescence measurements were made at7° temporal to the fovea and at the fovea in 30 normal subjects and in 3 selected patients.Topographic measurements were made in 3 subjects. Ex vivo measurements of fluorescenceof human retinal pigment epithelium (RPE) were obtained and compared to in vivo data.Results. Fundus fluorescence reveals a broad band of emission between 500 and 750 nm, amaximum of approximately 630 nm, and optimal excitation of approximately 510 nm. Exhib-iting a significant increase with age, this fluorescence is highest at 7° to 15° from the fovea,shows a well-defined foveal minimum, and decreases toward the periphery. In vivo fluores-cence spectra are consistent with those obtained ex vivo on human RPE. Measurements withshort wavelength excitation are strongly influenced by ocular media absorption and revealan additional minor fluorophore in the fovea.Conclusions. Spectral characteristics, correlation with age, topographic distribution, and retinallocation between the choriocapillaris and the photoreceptors suggest that the dominantfundus fluorophore is RPE lipofuscin. The mino fluorophorer is probably in the neurosensoryretina but has not been identified. Invest Ophthalmol Vis Sci. 1995;36:718-729.Ajipofuscin is an autofluorescent pigment that accu-mulates in the lysosomes of most aging eukaryoticcells. Intensively studied as a biomarker for cellularaging and as a cumulative index of oxidative damage,mounting evidence across the biologic spectrum indi-cates that lipofuscin may play an important role incellular aging and disease.In the human retinal pigment epithelium (RPE),lipofuscin accumulates as a byproduct of phagocytosisof photoreceptor outer segments. In advanced age,lipofuscin is a major constituent of the RPE cell be-

In Vivo Fluorescence of the Ocular Fundus Exhibits Retinal Pigment Epithelium Lipofuscin Characteristics

https://www.cell.com/biophysj/pdf/S0006-3495(02)75621-X.pdf

Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein

Fluorescence Spectroscopy of Neoplastic and Non-Neoplastic Tissues

Biochemical integrity of the brain is vital for normal functioning of the central nervous system (CNS). One of the factors contributing to cerebral biochemical impairment is a chemical process called oxidative stress. Oxidative stress occurs upon excessive free radical production resulting from an insufficiency of the counteracting antioxidant response system. The brain, with its high oxygen consumption and lipid-rich content, is highly susceptible to oxidative stress. Therefore, oxidative stress–induced damage to the brain has a strong potential to negatively impact normal CNS functions. Although oxidative stress has historically been considered to be involved mainly in neurodegenerative disorders such as Alzheimer disease, Huntington disease, and Parkinson disease, its involvement in neuropsychiatric disorders, including anxiety disorders and depression, is beginning to be recognized. This review is a discussion of the relevance of cerebral oxidative stress to impairment of emotional and mental well-being.

https://jpet.aspetjournals.org/content/jpet/360/1/201.full.pdf

R. Oshino

Papers

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.