Optical coherence tomography (OCT) is a powerful tool in ophthalmology that provides in vivo morphology of the retinal layers and their light scattering properties. The directional (angular) reflectivity of the retinal layers was investigated with focus on the scattering from retinal pigment epithelium (RPE). The directional scattering of the RPE was studied in three mice strains with three distinct melanin concentrations: albino (BALB/c), agouti (129S1/SvlmJ), and strongly pigmented (C57BL/6J). The backscattering signal strength was measured with a directional OCT system in which the pupil entry position of the narrow OCT beam can be varied across the dilated pupil of the eyes of the mice. The directional reflectivity of other retinal melanin-free layers, including the internal and external limiting membranes, and Bruch's membrane (albinos) were also measured and compared between the strains. The intensity of light backscattered from these layers was found highly sensitive to the angle of illumination, whereas the inner/outer segment (IS/OS) junctions showed a reduced sensitivity. The reflections from the RPE are largely insensitive in highly pigmented mice. The differences in directional scattering between strains shows that directionality decreases with an increase in melanin concentrations in RPE, suggesting increasing contribution of Mie scattering by melanosomes.

It has been recently demonstrated that structures corresponding to the cell bodies of highly transparent cells in the retinal ganglion cell layer could be visualized noninvasively in the living human eye by optical coherence tomography (OCT) via temporal averaging. Inspired by this development, we explored the application of volumetric temporal averaging in mice, which are important models for studying human retinal diseases and therapeutic interventions. A general framework of temporal speckle-averaging (TSA) of OCT and optical coherence tomography angiography (OCTA) is presented and applied to mouse retinal volumetric data. Based on the image analysis, the eyes of mice under anesthesia exhibit only minor motions, corresponding to lateral displacements of a few micrometers and rotations of a fraction of 1 deg. Moreover, due to reduced eye movements under anesthesia, there is a negligible amount of motion artifacts within the volumes that need to be corrected to achieve volume coregistration. In addition, the relatively good optical quality of the mouse ocular media allows for cellular-resolution imaging without adaptive optics (AO), greatly simplifying the experimental system, making the proposed framework feasible for large studies. The TSA OCT and TSA OCTA results provide rich information about new structures previously not visualized in living mice with non-AO-OCT. The mechanism of TSA relies on improving signal-to-noise ratio as well as efficient suppression of speckle contrast due to temporal decorrelation of the speckle patterns, enabling full utilization of the high volumetric resolution offered by OCT and OCTA. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

For in vivo mouse retinal imaging, especially with Adaptive Optics instruments, application of a contact lens is desirable, as it allows maintenance of cornea hydration and helps to prevent cataract formation during lengthy imaging sessions. However, since the refractive elements of the eye (cornea and lens) serve as the objective for most in vivo retinal imaging systems, the use of a contact lens, even with 0 Dpt. refractive power, can alter the system's optical properties. In this investigation we examined the effective focal length change and the aberrations that arise from use of a contact lens. First, focal length changes were simulated with a Zemax mouse eye model. Then ocular aberrations with and without a 0 Dpt. contact lens were measured with a Shack-Hartmann wavefront sensor (SHWS) in a customized AO-SLO system. Total RMS wavefront errors were measured for two groups of mice (14-month, and 2.5-month-old), decomposed into 66 Zernike aberration terms, and compared. These data revealed that vertical coma and spherical aberrations were increased with use of a contact lens in our system. Based on the ocular wavefront data we evaluated the effect of the contact lens on the imaging system performance as a function of the pupil size. Both RMS error and Strehl ratios were quantified for the two groups of mice, with and without contact lenses, and for different input beam sizes. These results provide information for determining optimum pupil size for retinal imaging without adaptive optics, and raise critical issues for design of mouse optical imaging systems that incorporate contact lenses.

Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4−/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4−/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.

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