In vivo measurement of time-resolved autofluorescence at the human fundus

Schweitzer, Dietrich; Hammer, Martin; Schweitzer, Frank; Anders, R.; Doebbecke, Torsten; Schenke, S.; Gaillard, Elizabeth R.

doi:10.1117/1.1806833

Cited by 132 publications

(120 citation statements)

References 25 publications

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“…The lifetimes and relative contributions of free and proteinbound FAD measured in vivo in normal tissues are consistent with those reported in solutions (11,29) and in the eye (30). The protein-bound FAD lifetime increased and the relative contribution of protein-bound FAD decreased with high-grade precancer only (Fig.…”

Section: Discussionsupporting

confidence: 77%

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

Skala

Riching

Gendron‐Fitzpatrick

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

915

901

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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...

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Section: Discussionsupporting

confidence: 77%

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

Skala

Riching

Gendron‐Fitzpatrick

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

915

901

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“…An offline registration of recorded images was introduced in 2002 (Schweitzer et al, 2002) and first clinical experiments in patients with age-related macular degeneration (AMD) were published in 2003 using a picosecond diode laser as light source (Schweitzer et al, 2003). Although the resolution was still low due to limited memory of the TCSPC electronics (64 Â 64 pixels with a size of 80 Â 80 mm 2 ), the images clearly revealed an extension of lifetimes in age-related macular degeneration (Schweitzer et al, 2004). In vitro and histological studies were performed to identify the fluorophores seen in fundus autofluorescence and to measure their emission spectra as well as lifetimes (Schweitzer et al, 2007a;Schweitzer et al, 2007b).…”

Section: Historical Backgroundmentioning

confidence: 99%

Fluorescence lifetime imaging ophthalmoscopy

Dysli

Wolf

Zinkernagel

2017

Acta Ophthalmologica

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a b s t r a c tImaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases.Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review.

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“…He found that lipofuscin-based methods using only one excitation wavelength (460 nm) would overestimate the MP levels by 0. 22 34 Human RPE contains the age pigment lipofuscin, which accumulates in the lysosomal body of the RPE cells. 35 Two components of lipofuscin exist that absorb strongly in the blue wavelength region and emit strongly in the orange-red region.…”

Section: Spectroscopymentioning

confidence: 99%

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

2006

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We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the bluegreen wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

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In vivo measurement of time-resolved autofluorescence at the human fundus

Cited by 132 publications

References 25 publications

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

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

Fluorescence lifetime imaging ophthalmoscopy

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

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