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This study aims to correlate adaptive optics-transscleral flood illumination (AO-TFI) images of the retinal pigment epithelium (RPE) in central serous chorioretinopathy (CSCR) with standard clinical images and compare cell morphological features with those of healthy eyes. After stitching 125 AO-TFI images acquired in CSCR eyes (including 6 active CSCR, 15 resolved CSCR, and 3 from healthy contralateral), 24 montages were correlated with blue-autofluorescence, infrared and optical coherence tomography images. All 68 AO-TFI images acquired in pathological areas exhibited significant RPE contrast changes. Among the 52 healthy areas in clinical images, AO-TFI revealed a normal RPE mosaic in 62% of the images and an altered RPE pattern in 38% of the images. Morphological features of the RPE cells were quantified in 54 AO-TFI images depicting clinically normal areas (from 12 CSCR eyes). Comparison with data from 149 AO-TFI images acquired in 33 healthy eyes revealed significantly increased morphological heterogeneity. In CSCR, AO-TFI not only enabled high-resolution imaging of outer retinal alterations, but also revealed RPE abnormalities undetectable by all other imaging modalities. Further studies are required to estimate the prognosis value of these abnormalities. Imaging of the RPE using AO-TFI holds great promise for improving our understanding of the CSCR pathogenesis., (© 2024. The Author(s).)

Objective: To image healthy retinal pigment epithelial (RPE) cells in vivo using Transscleral OPtical Imaging (TOPI) and to analyze statistics of RPE cell features as a function of age, axial length (AL), and eccentricity., Design: Single-center, exploratory, prospective, and descriptive clinical study., Participants: Forty-nine eyes (AL: 24.03 ± 0.93 mm; range: 21.9-26.7 mm) from 29 participants aged 21 to 70 years (37.1 ± 13.3 years; 19 men, 10 women)., Methods: Retinal images, including fundus photography and spectral-domain OCT, AL, and refractive error measurements were collected at baseline. For each eye, 6 high-resolution RPE images were acquired using TOPI at different locations, one of them being imaged 5 times to evaluate the repeatability of the method. Follow-up ophthalmic examination was repeated 1 to 3 weeks after TOPI to assess safety. Retinal pigment epithelial images were analyzed with a custom automated software to extract cell parameters. Statistical analysis of the selected high-contrast images included calculation of coefficient of variation (CoV) for each feature at each repetition and Spearman and Mann-Whitney tests to investigate the relationship between cell features and eye and subject characteristics., Main Outcome Measures: Retinal pigment epithelial cell features: density, area, center-to-center spacing, number of neighbors, circularity, elongation, solidity, and border distance CoV., Results: Macular RPE cell features were extracted from TOPI images at an eccentricity of 1.6° to 16.3° from the fovea. For each feature, the mean CoV was < 4%. Spearman test showed correlation within RPE cell features. In the perifovea, the region in which images were selected for all participants, longer AL significantly correlated with decreased RPE cell density (R Spearman, Rs = -0.746; P < 0.0001) and increased cell area (Rs = 0.668; P < 0.0001), without morphologic changes. Aging was also significantly correlated with decreased RPE density (Rs = -0.391; P  = 0.036) and increased cell area (Rs = 0.454; P  = 0.013). Lower circular, less symmetric, more elongated, and larger cells were observed in those > 50 years., Conclusions: The TOPI technology imaged RPE cells in vivo with a repeatability of < 4% for the CoV and was used to analyze the influence of physiologic factors on RPE cell morphometry in the perifovea of healthy volunteers., Financial Disclosures: Proprietary or commercial disclosure may be found after the references., (© 2022 by the American Academy of Ophthalmology. Published by Elsevier Inc.)

Shifted Excitation Raman Difference Spectroscopy (SERDS) is a non-destructive chemical analysis method capable of removing the fluorescence background and other disturbances from the Raman spectrum, thanks to the independence of the fluorescence with respect to the small difference in excitation wavelength. The spectrum difference is computed in a post-processing step. Here, we demonstrate the use of a lock-in camera to obtain an on-line analog SERDS spectra allowing longer exposure times and no saturation, leading to an improved Signal-to-Noise Ratio (SNR) and reduced data storage. Two configurations are presented: the first one uses a single laser and can remove excitation-independent disturbances, such as ambient light; the second employs two-wavelength shifted sources and removes fluorescence background similarly to SERDS. In both cases, we experimentally extrapolate the expected SNR improvement.