Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: To demonstrate the first near-infrared adaptive optics fluorescence lifetime imaging ophthalmoscopy (NIR-AOFLIO) measurements in vivo of the human retinal pigment epithelial (RPE) cellular mosaic and to visualize lifetime changes at different retinal eccentricities. Methods: NIR reflectance and autofluorescence were captured using a custom adaptive optics scanning light ophthalmoscope in 10 healthy subjects (23-64 years old) at seven eccentricities and in two eyes with retinal abnormalities. Repeatability was assessed across two visits up to 8 weeks apart. Endogenous retinal fluorophores and hydrophobic whole retinal extracts of Abca4-/- pigmented and albino mice were imaged to probe the fluorescence origin of NIR-AOFLIO. Results: The RPE mosaic was resolved at all locations in five of seven younger subjects (<35 years old). The mean lifetime across near-peripheral regions (8° and 12°) was longer compared to near-foveal regions (0° and 2°). Repeatability across two visits showed moderate to excellent correlation (intraclass correlation: 0.88 [τm], 0.75 [τ1], 0.65 [τ2], 0.98 [a1]). The mean lifetime across drusen-containing eyes was longer than in age-matched healthy eyes. Fluorescence was observed in only the extracts from pigmented Abca4-/- mouse. Conclusions: NIR-AOFLIO was repeatable and allowed visualization of the RPE cellular mosaic. An observed signal in only the pigmented mouse extract infers the fluorescence signal originates predominantly from melanin. Variations observed across the retina with intermediate age-related macular degeneration suggest NIR-AOFLIO may act as a functional measure of a biomarker for in vivo monitoring of early alterations in retinal health.

Fluorescence Lifetime Imaging of Human Retinal Pigment Epithelium in Pentosan Polysulfate Toxicity Using Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: Fluorescence lifetime ophthalmoscopy (FLIO) is an emerging clinical modality that could provide biomarkers of retinal health beyond fluorescence intensity. Adaptive optics (AO) ophthalmoscopy provides the confocality to measure fluorescence lifetime (FL) primarily from the retinal pigment epithelium (RPE) whereas clinical FLIO has greater influence from fluorophores in the inner retina and lens. Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) measures of FL in vivo could provide insight into RPE health at different stages of disease. In this study, we assess changes in pentosan polysulfate sodium (PPS) toxicity, a recently described toxicity that has clinical findings similar to advanced age-related macular degeneration. Methods: AOFLIO was performed on three subjects with PPS toxicity (57-67 years old) and six age-matched controls (50-64 years old). FL was analyzed with a double exponential decay curve fit and with phasor analysis. Regions of interest (ROIs) were subcategorized based on retinal features on optical coherence tomography (OCT) and compared to age-matched controls. Results: Twelve ROIs from PPS toxicity subjects met the threshold for analysis by curve fitting and 15 ROIs met the threshold for phasor analysis. Subjects with PPS toxicity had prolonged FL compared to age-matched controls. ROIs of RPE degeneration had the longest FLs, with individual pixels extending longer than 900 ps. Conclusions: Our study shows evidence that AOFLIO can provide meaningful information in outer retinal disease beyond what is obtainable from fluorescence intensity alone. More studies are needed to determine the prognostic value of AOFLIO.

Spectral Dependence of Light Exposure on Retinal Pigment Epithelium Disruption in Living Primate Retina.

Purpose: RPE disruption with light exposures below or close to the American National Standards Institute (ANSI) photochemical maximum permissible exposure (MPE) have been observed, but these findings were limited to two wavelengths. We have extended the measurements across the visible spectrum. Methods: Retinal imaging with fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) was used to provide an in vivo measure of RPE disruption at a cellular level. The threshold retinal radiant exposures (RREs) for RPE disruption (localized detectable change in the fluorescence image) were determined at 460, 476, 488, 530, 543, 561, 594, 632, and 671 nm (uniform 0.5° square exposure) using multiples locations in 4 macaques. Results: FAOSLO is sensitive in detecting RPE disruption. The visible light action spectrum dependence for RPE disruption with continuous wave (CW) extended field exposures was determined. It has a shallower slope than the current ANSI blue-light hazard MPE. At all wavelengths beyond 530 nm, the disruption threshold is below the ANSI blue-light hazard MPE. There is reciprocity of exposure irradiance and duration for exposures at 460 and 594 nm. Conclusions: We measured with FAOSLO the action spectrum dependence for photochemical RPE disruption across the visible light spectrum. Using this in vivo measure of phototoxicity provided by FAOSLO, we find that thresholds are lower than previously measured. The wavelength dependence in our data is considerably shallower than the spectral dependence of the traditional ANSI blue-light hazard, emphasizing the need for more caution with increasing wavelength than expected.

Erratum: Adaptive optics fluorescence lifetime imaging ophthalmoscopy of in vivo human retinal pigment epithelium: erratum.

[This corrects the article on p. 1737 in vol. 13, PMID: 35414970.].

Separate lifetime signatures of macaque S cones, M/L cones, and rods observed with adaptive optics fluorescence lifetime ophthalmoscopy.

In the retina, several molecules involved in metabolism, the visual cycle, and other roles exhibit intrinsic fluorescence. The overall properties of retinal fluorescence depend on changes to the composition of these molecules and their environmental interactions due to transient functional shifts, especially in disease. This behooves the understanding of the origins and deviations of these properties within the multilayered retina at high lateral and axial resolution. Of particular interest is the fluorescence lifetime, a potential biomarker of function and disease independent of fluorescence intensity that can be measured in the retina with adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO). This work demonstrates the utility of the phasor method of analysis, an alternate approach to traditional multiexponential fitting, to evaluate photoreceptor two-photon excited AOFLIO data and separate them based on functional differences. Phasor analysis on fluorescence lifetime decay data allowed the repeatable segregation of S from M/L cones, likely from differences in functional or metabolic demands. Furthermore, it is possible to track the lifetime changes in S cones after photodamage. Phasor analysis increases the sensitivity of AOFLIO to functional differences between cells and has the potential to improve our understanding of pathways involved in normal and diseased conditions at the cellular scale throughout the retina.

Adaptive optics fluorescence lifetime imaging ophthalmoscopy of in vivo human retinal pigment epithelium.

The intrinsic fluorescence properties of lipofuscin - naturally occurring granules that accumulate in the retinal pigment epithelium - are a potential biomarker for the health of the eye. A new modality is described here which combines adaptive optics technology with fluorescence lifetime detection, allowing for the investigation of functional and compositional differences within the eye and between subjects. This new adaptive optics fluorescence lifetime imaging ophthalmoscope was demonstrated in 6 subjects. Repeated measurements between visits had a minimum intraclass correlation coefficient of 0.59 Although the light levels were well below maximum permissible exposures, the safety of the imaging paradigm was tested using clinical measures; no concerns were raised. This new technology allows for in vivo adaptive optics fluorescence lifetime imaging of the human RPE mosaic.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of photoreceptors and retinal pigment epithelium in the living non-human primate eye.

Fluorescence lifetime imaging has demonstrated promise as a quantitative measure of cell health. Adaptive optics two-photon excited fluorescence (TPEF) ophthalmoscopy enables excitation of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle, providing in vivo visualization of retinal structure and function at the cellular scale. Combining these technologies revealed that macaque cones had a significantly longer mean TPEF lifetime than rods at 730 nm excitation. At 900 nm excitation, macaque photoreceptors had a significantly longer mean TPEF lifetime than the retinal pigment epithelium layer. AOFLIO can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes.

Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics.

Purpose: The development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina. Methods: In the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells. Results: This method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved. Conclusions: Although different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates. Translational Relevance: With this model, a variety of vision restoration methods can be tested in the non-human primate.

Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution.

Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX+/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.

Imaging Retinal Activity in the Living Eye.

Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons. In some cases, the methods have existed for years and are still being optimized. In others, new methods such as optophysiology are revealing novel patterns of retinal function in animal models that have the potential to change our understanding of the functional capacity of the retina. Together, the advances in retinal imaging mark an important milestone that shifts attention away from anatomy alone and begins to probe the function of healthy and diseased eyes.

Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

Selective S Cone Damage and Retinal Remodeling Following Intense Ultrashort Pulse Laser Exposures in the Near-Infrared.

Purpose: Infrared ultrashort pulse lasers are becoming increasingly popular for applications in the living eye. However, safety standards are not yet well established. Here we investigate retinal damage close to threshold for this pulse regime in the living macaque eye. Methods: Retinal radiant exposures between 214 and 856 J/cm2 were delivered to the photoreceptor layer with an ultrashort pulse laser (730 nm, 55 fs, 80 MHz) through a two-photon adaptive optics scanning light ophthalmoscope. Retinal exposures were followed up immediately after and over several weeks with high-resolution reflectance and two-photon excited fluorescence ophthalmoscopy, providing structural and functional information. Results: Retinal radiant exposures of 856 J/cm2 resulted in permanent S cone damage. Immediately after the exposure, the affected cones emitted about 2.6 times less two-photon excited fluorescence (TPEF) and showed an altered TPEF time course. Several weeks after the initial exposure, S cone outer and inner segments had disappeared. The space was filled by rods in the peripheral retina and cones near the fovea. Conclusion: Interestingly, S cones are the receptor class with the lowest sensitivity in the near-infrared but are known to be particularly susceptible to ultraviolet and blue light. This effect of selective S cone damage after intense infrared ultrashort pulse laser exposure may be due to nonlinear absorption and distinct from pure thermal and mechanical mechanisms often associated with ultrashort pulse lasers.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Imaging individual neurons in the retinal ganglion cell layer of the living eye.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Formation and Clearance of All-Trans-Retinol in Rods Investigated in the Living Primate Eye With Two-Photon Ophthalmoscopy.

Purpose: Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol. Methods: Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics. Results: Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds. Conclusions: This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.

Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans.

Two-photon ophthalmoscopy has potential for in vivo assessment of function of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.

Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye.

PURPOSE: Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. METHODS: Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. RESULTS: All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. CONCLUSIONS: This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.

In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones.

PURPOSE: The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. METHODS: We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye's pupil. RESULTS: The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. CONCLUSIONS: Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas.

Cone and rod loss in Stargardt disease revealed by adaptive optics scanning light ophthalmoscopy.

IMPORTANCE: Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy. OBSERVATIONS: Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P < .001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations. CONCLUSIONS AND RELEVANCE: This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1.