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.

Foveal Retinal Ganglion Cells Develop Altered Calcium Dynamics Weeks After Photoreceptor Ablation.

Purpose: Physiological changes in retinal ganglion cells (RGCs) have been reported in rodent models of photoreceptor (PR) loss, but this has not been investigated in primates. By expressing both a calcium indicator (GCaMP6s) and an optogenetic actuator (ChrimsonR) in foveal RGCs of the macaque, we reactivated RGCs in vivo and assessed their response in the weeks and years after PR loss. Design: We used an in vivo calcium imaging approach to record optogenetically evoked activity in deafferented RGCs in primate fovea. Cellular scale recordings were made longitudinally over a 10-week period after PR ablation and compared with responses from RGCs that had lost PR input >2 years prior. Participants: Three eyes received PR ablation, the right eye of a male Macaca mulatta (M1), the left eye of a female Macaca fascicularis (M2), and the right eye of a male Macaca fascicularis (M3). Two animals were used for in vivo recording, 1 for histological assessment. Methods: Cones were ablated with an ultrafast laser delivered through an adaptive optics scanning light ophthalmoscope (AOSLO). A 0.5 second pulse of 25 Hz 660 nm light optogenetically stimulated RGCs, and the resulting GCaMP fluorescence signal was recorded using an AOSLO. Measurements were repeated over 10 weeks immediately after PR ablation, at 2.3 years and in control RGCs. Main Outcome Measures: The calcium rise time, decay constant, and sensitivity index of optogenetic-mediated RGC were derived from GCaMP fluorescence recordings from 221 RGCs (animal M1) and 218 RGCs (animal M2) in vivo. Results: After PR ablation, the mean decay constant of the calcium response in RGCs decreased 1.5-fold (standard deviation 1.6 ± 0.5 seconds to 0.6 ± 0.3 seconds) over the 10-week observation period in subject 1 and 2.1-fold (standard deviation 2.5 ± 0.5 seconds to 1.2 ± 0.2 seconds) within 8 weeks in subject 2. Calcium rise time and sensitivity index were stable. Optogenetic reactivation remained possible 2.3 years after PR ablation. Conclusions: Altered calcium dynamics developed in primate foveal RGCs in the weeks after PR ablation. The mean decay constant of optogenetic-mediated calcium responses decreased 1.5- to twofold. This is the first report of this phenomenon in primate retina and further work is required to understand the role these changes play in cell survival and activity. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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.

Foveal RGCs develop altered calcium dynamics weeks after photoreceptor ablation.

Objective or purpose: Physiological changes in retinal ganglion cells (RGCs) have been reported in rodent models of photoreceptor (PR) loss but this has not been investigated in primates. By expressing both a calcium indicator (GCaMP6s) and an optogenetic actuator (ChrimsonR) in foveal RGCs of the macaque, we reactivated RGCs in vivo and assessed their response in the weeks and years following PR loss. Design: We used an in vivo calcium imaging approach to record optogenetically evoked activity in deafferented RGCs in primate fovea. Cellular scale recordings were made longitudinally over a 10 week period following photoreceptor ablation and compared to responses from RGCs that had lost photoreceptor input more than two years prior. Participants: Three eyes received photoreceptor ablation, OD of a male Macaca mulatta (M1), OS of a female Macaca fascicularis (M2) and OD of a male Macaca fascicularis (M3). Two animals were used for in vivo recording, one for histological assessment. Methods: Cones were ablated with an ultrafast laser delivered through an adaptive optics scanning light ophthalmoscope (AOSLO). A 0.5 s pulse of 25Hz 660nm light optogenetically stimulated RGCs, and the resulting GCaMP fluorescence signal was recorded using AOSLO. Measurements were repeated over 10 weeks immediately after PR ablation, at 2.3 years and in control RGCs. Main Outcome measures: The calcium rise time, decay constant and sensitivity index of optogenetic mediated RGC were derived from GCaMP fluorescence recordings from 221 RGCs (Animal M1) and 218 RGCs (Animal M2) in vivo. Results: Following photoreceptor ablation, the mean decay constant of the calcium response in RGCs decreased 1.5 fold (1.6±0.5 s to 0.6±0.3 s SD) over the 10 week observation period in subject 1 and 2.1 fold (2.5±0.5 s to 1.2±0.2 s SD) within 8 weeks in subject 2. Calcium rise time and sensitivity index were stable. Optogenetic reactivation remained possible 2.3 years after PR ablation. Conclusions: Altered calcium dynamics developed in primate foveal RGCs in the weeks after photoreceptor ablation. The mean decay constant of optogenetic mediated calcium responses decreased 1.5 - 2-fold. This is the first report of this phenomenon in primate retina and further work is required to understand the role these changes play in cell survival and activity.

Contributed Session II: In vivo imaging of immune cell activity in primate retina after photoreceptor ablation.

The non-human primate (NHP) is the gold standard animal model for preclinical development of gene and cell based therapies for vision restoration. However, the ocular immune response to these interventions remains poorly understood. We conducted a proof of concept study using offset aperture adaptive optics scanning light ophthalmoscopy (AOSLO) to visualize cellular-scale changes in the primate retina following photoreceptor (PR) ablation. Ultrafast 730nm laser exposure at 26.6 - 32.5 J/cm2 was used to create six lesions in four NHPs. Offset aperture images focused on retinal vascular layers were collected with an offset distance of ~10 Airy Disk Diameters from 15 minutes up to three hours after PR ablation. We observed putative immune cells in and around vessels supplying the lesioned areas. Consistent with previous findings in murine models, cells within vessels adhered to the inner wall, exhibited crawling behavior, and had a diameter ranging from ~9.3 - 11.5 µm. Additionally, we observed the emergence of cellular-scale structures above the PR layer that originated in the center of the lesion 15 minutes post-insult and gradually radiated outward. Vascular perfusion was maintained in these regions. Our data suggest that offset aperture imaging offers cellular-scale, label free, in vivo assessment of the retinal response to insult in NHPs and could be employed to advance our understanding of the ocular immune response provoked by disease and therapeutic interventions.

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.].

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.

Optogenetic therapy restores retinal activity in primate for at least a year following photoreceptor ablation.

All retina-based vision restoration approaches rely on the assumption that photoreceptor loss does not preclude reactivation of the remaining retinal architecture. Whether extended periods of vision loss limit the efficacy of restorative therapies at the retinal level is unknown. We examined long-term changes in optogenetic responsivity of foveal retinal ganglion cells (RGCs) in non-human primates following localized photoreceptor ablation by high-intensity laser exposure. By performing fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) of RGCs expressing both the calcium indicator GCaMP6s and the optogenetic actuator ChrimsonR, it was possible to track optogenetic-mediated calcium responses in deafferented RGCs over time. Fluorescence fundus photography revealed a 40% reduction in ChrimsonR fluorescence from RGCs lacking photoreceptor input over the 3 weeks following photoreceptor ablation. Despite this, in vivo imaging revealed good cellular preservation of RGCs 3 months after the loss of photoreceptor input, and histology confirmed good structural preservation at 2 years. Optogenetic responses of RGCs in primate persisted for at least 1 year after the loss of photoreceptor input, with a sensitivity index similar to optogenetic responses recorded in intact retina. These results are promising for all potential therapeutic approaches to vision restoration that rely on preservation and reactivation of RGCs.

Diffraction efficiency performance of random anti-reflecting subwavelength surface structures on prefabricated fused silica binary gratings.

Random anti-reflecting subwavelength surface structures have been reported to enhance transmission of optical windows and lenses. Specifically, for fused silica substrates, 99.9% specular transmission has been verified by various groups. Diffractive optical elements, such as gratings, also experience net Fresnel losses on both their planar and structured surfaces. We investigated the performance of prefabricated 50% duty-cycle, binary, fused silica linear gratings, with a period of 1.6 µm, before and after application of random anti-reflecting subwavelength surface structures, in order to reduce their initial Fresnel reflectivity. We compared the diffraction order directions and their efficiencies at three test wavelengths: 594, 612, and 633 nm, for both TE(s) and TM(p) incident light polarization states, under three different mountings: normal, first Bragg, and second Bragg incidence. We report transmission enhancement of the sum of all propagating grating orders for all cases tested by factors between 2% and 10%, with reduction of the respective reflected orders by similar ratios. Transmission enhancement of the -2 diffraction order at Bragg incidence suggests that the random etch has different rates between the raised and lowered linear grating topography.