The video demonstrates the procedure for ex vivo color fundus photography, detailing the setup of the camera and light sources, preparation of the eye globe in a quartz crucible, and visualization techniques to observe the interior ocular fundus using a stereomicroscope. Proper orientation and positioning of anatomical landmarks are emphasized.

preparation for ex vivo color fundus photography

OCT-basierte multimodale Bildgebung von menschlichen Spenderaugen für die AMD-Forschung

Fifteen years of managing neovascular age-related macular degeneration (AMD) with anti-VEGF therapy under the guidance of optical coherence tomography (OCT) has offered new insights into the progression sequence and microarchitecture of this prevalent cause of vision loss. A key recognition is that AMD is a three-dimensional disease involving the neurosensory retina, retinal pigment epithelium (RPE), and choroid. As a result of the OCT imaging of trial patients and the fellow eyes of treated clinic patients, the features of pathology beyond those seen by color fundus photography, a clinical standard for decades, are now recognized. These include intraretinal neovascularization (type 3 macular neovascularization1, formerly angiomatous proliferation), subretinal drusenoid deposits (SDDs, also called reticular pseudodrusen)2, multiple pathways of RPE fate3,4, and intensely gliotic M&#252;ller cells in atrophy5,6.
Model systems lacking maculas (cells and animals) recreate some slices of this complex disease7,8,9. Further success in ameliorating the burden of AMD could come from the discovery and exploration of primary pathology in human eyes, understanding the unique cellular composition of the macula, followed by translation to model systems. This report portrays a three decade collaboration between an academic research laboratory and an eye bank. The goals of the tissue characterization methods described herein are two-fold: 1) to inform evolving diagnostic technology by demonstrating the basis of fundus appearance and imaging signal sources with microscopy, and 2) to classify AMD specimens for targeted (immunohistochemistry) and untargeted molecular discovery techniques (imaging mass spectrometry, IMS, and spatial transcriptomics) that preserve the cone-only fovea and rod-rich para- and perifovea. Such studies could accelerate the translation to clinical OCT, for which a progression sequence and longitudinal follow-up are possible through eye-tracking. This technology, which is designed to monitor treatment effects, registers scans from one clinic visit to the next using retinal vessels. Linking eye-tracked OCT to laboratory results obtained with destructive techniques could provide a new level of prognostic value to molecular findings.
In 1993, the research laboratory captured color photographs of postmortem fundus on film10. This effort was inspired by the superb photomicroscopy and histology of the human peripheral retina by Foos and colleagues11,12,13&#160;and the extensive AMD clinicopathologic correlations by Sarks et al.14,15. Starting in 2009, ex vivo multimodal imaging (MMI) anchored on spectral domain OCT was adopted. This transition was inspired by the similar efforts of others16,17&#160;and especially by the realization that so much of the ultrastructure described by the Sarks was available in three dimensions, over time, in the clinic18,19. The goal was to acquire eyes with attached maculas in a reasonable time frame for well-powered studies of cellular-level phenotypes in the retina, RPE, and choroid. The intent was to move beyond &#34;per eye&#34; statistics to &#34;per lesion type,&#34; a standard influenced by the &#34;vulnerable plaque&#34; concepts from cardiovascular disease20,21.
The protocol in this report reflects experience with nearly 400 pairs of donor eyes accessioned in several streams. In 2011-2014, the Project MACULA website of AMD histopathology was created, which includes layer thicknesses and annotations from 142 archived specimens. These eyes were preserved from 1996-2012 in a glutaraldehyde-paraformaldehyde fixative for high-resolution epoxy-resin histology and electron microscopy. All the fundi had been photographed in color when received and were reimaged by OCT just prior to histology. An eye holder originally designed for optic nerve studies22&#160;was used to accommodate an 8 mm diameter full-thickness tissue punch centered on the fovea. OCT B-scans through the foveal center and a site 2 mm superior, corresponding to histology at the same levels, were uploaded to the website, plus a color fundus photograph. The choice of the OCT planes was dictated by the prominence of AMD pathology under the fovea23&#160;and the prominence of SDDs in rod-rich areas superior to the fovea24,25.
Starting in 2013, eyes imaged with OCT-anchored MMI during life were available for direct clinicopathologic correlations. Most (7 of 10 donors) involved patients at a retina referral practice (author: K.B.F.), which offered an advanced directive registry for patients interested in donating their eyes after death for research purposes. The eyes were recovered and preserved by the local eye bank, transferred to the laboratory, and prepared in the same way as the Project MACULA eyes. Pre-mortem clinical OCT volumes were seamlessly read in the laboratory, thus aligning the pathology features seen during life with the features seen under the microscope26.
Starting in 2014, prospective eye collection began by screening for AMD in donor eyes without a clinical history but preserved during a defined time limit (6 h). For this purpose, the eye holder was modified to accommodate a whole globe. This reduced the chance of detachment around the cut edges of the previously used 8 mm punch. The eyes were preserved in 4% buffered paraformaldehyde for immunohistochemistry and transferred to 1% the next day for long-term storage. In 2016-2017 (pre-pandemic), 184 eyes from 90 donors were recovered. The statistics and images in this report are generated from this series. During the pandemic era (2020 lockdowns and aftermath), prospective collections for transcriptomics and IMS collaborations continued at a reduced pace, essentially using the 2014 methods.
Other methods for donor eye assessment are available. The Minnesota Grading System (MGS)27,28&#160;is based on the AREDS clinical system for color fundus photography29. The limitations of this method include the combining of atrophic and neovascular AMD into one stage of &#34;late AMD&#34;. Further, the MGS entails the removal of the neurosensory retina before the photo-documentation of the RPE choroid. This step dislodges SDDs to varying degrees30,31&#160;and removes the spatial correspondence of the outer retina and its support system. Thus, efforts to link metabolic demand and signaling from the retina to pathology in the RPE-choroid may be impeded. The Utah System implemented MMI using ex vivo color photography and OCT to categorize eyes destined for dissection into regions for RNA and protein extractions32. Although preferable to whole eyecup extractions, the 3 mm diameter area at the highest risk for AMD progression33,34&#160;represents only 25% of a 6 mm diameter fovea-centered punch. Thus, techniques that can localize findings in reference to the fovea, such as serial sectioning for immunohistochemistry, are advantageous.

Autor im Rampenlicht: Ex-vivo-OCT-basierte multimodale Bildgebung von menschlichen Spenderaugen für die Erforschung der altersbedingten Makuladegeneration  

Ex Vivo OCT-basierte multimodale Bildgebung menschlicher Spenderaugen zur Erforschung der altersbedingten Makuladegeneration

Watch this Scientific Journal Video about Preparation for Ex Vivo Color Fundus Photography at JoVE.com

Preparation for Ex Vivo Color Fundus Photography   

Vorbereitung für die Ex-vivo-Farbfundusfotografie 

Um mit der Ex-vivo-Farbfundusfotografie zu beginnen, stellen Sie die hochauflösende Multimedia-Schnittstellenkamera auf die manuelle ISO-Funktion ein. Ordnen Sie zwei Lichtquellen senkrecht zueinander an und drehen Sie die Epi-Illuminations-Lichtquellen auf volle Leistung. Führen Sie am Präpariermikroskop die ein Millimeter große Rubinperle in den hinteren Pol ein.Als nächstes wird der hintere Pol des Augapfels in einen mit Puffer gefüllten 30-Milliliter-Quarztiegel eingeführt. Wenn das Gewebe absinkt, führen Sie eine Versteifung zwischen dem Auge und der Wand des Tiegels ein. Setzen Sie den Globus im Tiegel auf den Stereomikroskoptisch, um den inneren Augenhintergrund durch das Mikroskop mit der niedrigsten Vergrößerung zu beobachten.Richten Sie das Auge aus, indem Sie die Gewebemarkierung an der 12-Uhr-Position, den Sehnervenkopf und die Fovea fünf Grad unterhalb des Sehnervenkopfes identifizieren.

preparation-for-ex-vivo-color-fundus-photography

Research

JoVE Journal

Neuroscience

Preparation for Ex Vivo Color Fundus Photography

198 Aufrufe. Um mit der Ex-vivo-Farbfundusfotografie zu beginnen, stellen Sie die hochauflösende Multimedia-Schnittstellenkamera auf die manuelle ISO-Funktion ein. Ordnen Sie zwei Lichtquellen senkrecht zueinander an und drehen Sie die Epi-Illuminations-Lichtquellen auf volle Leistung. Führen Sie am Präpariermikroskop die ein Millimeter große Rubinperle in den hinteren Pol ein.Als nächstes wird der hintere Pol des Augapfels in einen mit Puffer gefüllten 30-Milliliter-Quarztiegel eingeführt. ...

Serie: Preparation for Ex Vivo Color Fundus Photography   

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...