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Laurenz Sonnentag, Salvatore Grisanti, Martin Rudolf; ex vivo Spectral Domain Optical Coherence Tomography (SD-OCT) of Retina-Choriocapillaris (R-CC) Specimen: Factors influencing Image Quality. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4147.
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SD-OCT enables high-resolution in vivo imaging of the retina, RPE and choriocapillaris. For better interpretation a direct correlation with histological sections is needed, for which scanned donor eyes are a potential source. In this study we evaluated parameters influencing the quality of R-CC histology samples and their ex vivo OCT scans.
Enucleated swine eyes were fixed at 6 different post mortem time points in 4 different fixatives: A: 4% Paraformaldehyde (PA); B: 4% PA/2% Glutaraldehyde (GA); C: 4% Formaldehyde (FA); D: 1% FA/1.25% GA. Isolated R-CC specimens were securely placed in our customized scanning-embedding (SE)-module, scanned by a Spectralis OCT (Heidelberg Ing., Germany), embedded without change in position and prepared for histology. Macaque eyes immediately fixed in 4% PA or 4% PA/2.5 % GA were handled accordingly. Quality of fixation and ex vivo OCT images was evaluated.
Both post mortem (pm) time and the fixatives have significant impact on the quality of ex vivo OCT scans and histology. Best histological results were achieved when samples were fixed within 3 hrs pm. Within 12 hrs the results were still acceptable. After this time retinal detachments dominated which minimized the samples value for further processing. OCT scans of specimens in fixatives A and C reached higher resolution where the identification of single retinal layers was better than those fixed in B and D containing GA. Specimens fixed with GA showed an increased OCT reflectivity, whereas their histologic sections had better morphological results. With our SE-module close matches of OCT images and histological sections of macaque maculae were achieved.
For optimal ex vivo OCT imaging and histology of R-CC specimen a short pm time until fixation is needed. Fixatives influence the quality of histology and OCT scans due to their different degree of protein cross-linking. Stronger fixation results in an increased reflectivity in OCT images but provides better morphology. These findings provide useful information for future histological studies which can improve the interpretation of clinical OCT scans by exposing the underlying pathologies.
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