The Imaging Core Unit (ICU) provides NEI intramural scientists access and training in a variety of high resolution imaging and analysis applications including confocal microscopy (Zeiss LSM 700, Zeiss LSM 780 and Olympus FV1000 laser scanning confocal microscope systems), total internal reflectance fluorescence ( Zeiss -TIRF), laser capture microdissection (Zeiss-PALM), ex vivo imaging of retina explants and stem cell cultures. A 2-photon microscope (Olympus Fluoview 1000 + Spectra Physics Mai Tai Deep See TiSapphire laser) has dramatically expand opportunities for imaging ocular tissues at greater depths and with reduced photodamage. A primary objective of the ICU is to pair state-of-the-art instrumentation with novel imaging approaches in order to leverage studies with the potential for significant clinical application. The ICU has developed improved methods for quantifying choroidal neovascularization in an experimental animal model. These studies have in turn lead to the identification of potent anti-angiogenic agents with the potential for retarding new blood vessel growth associated with the wet form of age-related macular degeneration.

During the past year, we continued a collaborative project with NEI Office of Scientific Information Officer (OISO) and BioTeam to develop automated segmentation of OCT images for retinal layers using machine-learning (ML) tools. We expanded our learning dataset to >1600 labelled images, and included both U-Net and Deeplabv3 model. The final product, AI-segmenter containing 12 independent models for segmentation of mouse OCT image. In addition, we also created a custom-made program to semi-automatically select the best segmented lines for the OCT images. Using AI-segmenter to segment and measure different retinal layer thickness of wildtype (C57BL/6J) mice, we noticed inferior / superior asymmetry in the thickness of outer nuclear layer (ONL). ONL of inferior retina is a few micron thicker than superior retina, suggesting inferior contains more photoreceptor cells than superior retina does. On the other hand, the length from external limiting membrane (ELM) to retinal pigment epithelium (RPE) is longer for superior retina than the length for inferior. As ELM-RPE length is mainly determined by rod photoreceptor inner and outer segment length, the asymmetry of ELM-RPE length suggest there are more rod photoreceptor in superior retina than in inferior retina. Based on these observations, asymmetry of ONL thickness reflects the concentration of cone photoreceptors in inferior mouse retina. To test our hypothesis, we examined RPE65 knockout mice and their littermate. It has been reported that cone photoreceptors degenerate first in this RPE65KO mouse model. We verified cone degeneration in 4-months old mice using whole-mount immunohistochemistry. For pigmented wildtype retina, there are more cone photoreceptors in inferior retina than those in superior retina. Degeneration of cone photoreceptors starts from inferior retina. Consequently, although both inferior and superior showed reduction in number of cone photoreceptors, inferior retina contains much less cones than superior retina for RPE65 KO mice. ONL thickness was measured from OCT images using AI-segmenter. For RPE65 KO mice, ONL thickness is thinner for inferior retina than the thickness of superior retina, whereas WT littermates showed thicker ONL for inferior retina than superior retina. These results support our hypothesis that asymmetry of ONL thickness is mediated by concentration of cone photoreceptors in the inferior mouse retina. In conclusion, our study showed that AI-segmenter could provide accurate measure of retinal layer thickness for mouse OCT image, with sensitivity of micron level. Inferior / superior retina asymmetry detected on OCT image provides a non-invasive measure of rod and cone photoreceptor distribution in mouse retina.

This study establishes a clinical database and biospecimen repository related to a variety of retinal conditions, particularly age-related macular degeneration and diabetic retinopathy. In secondary studies, these may be used for the identification of novel factors relevant to the pathogenesis, progression, and response to treatment of these retinal conditions. Objectives: This study provides for the standardized collection of longitudinal clinical data and for serial collection, processing, and storage of a variety of biospecimens. In secondary studies, the clinical data set and biospecimen repository may be used to identify novel genetic factors, biomarkers, and experimental models associated with pathogenesis, progression, and response to treatment for various conditions of the retina and their associated systemic correlates of disease. Study Population: We plan to accrue 200 participants with age-related macular degeneration (AMD), 125 participants with diabetic retinopathy, up to 200 participants with other retinal diseases, and 125 participants without any retinal disease. Design: This protocol specifies prospective collection of longitudinal clinical data related to multiple retinal diseases and suitable controls incorporating: 1. A standardized testing schedule; and 2. Collection of biospecimens for research purposes, for which sampling does not incur more than minimal risk to participants. In secondary studies, outcome Measures may include the interaction of key parameters of phenotype (such as visual acuity and retinal features on ocular imaging) with genetic variants and other biomarkers identified from biospecimens, and the characterization of new experimental models of eye health and disease. In the reporting period, we continued longitudinal testing and data collection for participants and enrolled a small number of additional participants.

RESEARCH PLAN: COMPUTATIONAL OPHTHALMOLOGY MODULE OVERVIEW At UCSD, structural imaging instruments such as the spectral domain optical coherence tomography (SD-OCT) and functional instruments are used extensively in animal (Drs. Weinreb, Freeman, Yu, La Spada, Zhang, Freeman) and human studies of glaucoma (Drs. Weinreb, Ju, Lindsay, Zangwill, Medeiros, Balasubramanian) and retinal disease (Drs. Freeman, Yu, Bartsch, Zhang, Cheng). The recent development and commercialization of imaging instruments such as SD-OCT has brought a significant improvement in our ability to visualize and measure the retina in-vivo in both animals and humans. These instruments have potential to dramatically improve our ability to understand the histopathology of major eyes diseases including glaucoma, age-related macular degeneration and diabetic retinopathy. These instruments represent a generational leap forward in technological development and several orders of magnitude more data than previous instruments. The challenge facing both ophthalmic clinicians and researchers is how best to utilize the vast quantity of data to 1) enhance our understanding of the histopathology of eye diseases, and 2) identify structural biomarkers of disease and its progression toward the ultimate goal of improving patient management. The computational ophthalmology module will provide essential centralized resources to support the computationally intensive analysis of structural imaging and functional tests used in animal and human vision research studies. This module will provide dedicated computational resources with a capacity to meet high computational demands and software toolkits that leverage these computational resources so that researchers can analyze the complex and data intensive retinal datasets (imaging and functional testing) outside of the proprietary software available with each ophthalmic test.

The Imaging Core Unit (ICU) provides NEI Intramural scientists access and training in a variety of high resolution imaging and analysis applications including confocal microscopy (Zeiss LSM 700, Zeiss LSM 880 Airy-Scan, Olympus FV1000 and Leica SP8 laser scanning confocal microscope systems).The facility provides expertise in ex vivo imaging of retina explants and stem cell cultures. In addition to confocal applications, the Leica SP8 Resonant scanning system utilizes a Spectra Physics Mai Tai "Deep-See" TiSapphire femptosecond laser which has dramatically expanded opportunities for imaging ocular tissues at greater depths and with reduced photo-damage. A primary objective of the ICU is to pair state-of-the-art instrumentation with novel imaging approaches in order to leverage studies with the potential for significant clinical application. The ICU has developed improved methods for quantifying choroidal neovascularization in an experimental animal model. These studies have in turn lead to the identification of potent anti-angiogenic agents with the potential for retarding new blood vessel growth associated with the wet form of age-related macular degeneration.