Subcellular Comparison of Visible-Light Optical Coherence Tomography and Electron Microscopy in the Mouse Outer Retina

Abstract

Purpose We employed in vivo, 1.0-µm axial resolution visible-light optical coherence tomography (OCT) and ex vivo electron microscopy (EM) to investigate three subcellular features in the mouse outer retina: reflectivity oscillations inner to band 1 (study 1); hyperreflective band 2, attributed to the ellipsoid zone or inner segment/outer segment (IS/OS) junction (study 2); and the hyperreflective retinal pigment epithelium (RPE) within band 4 (study 3). Methods Pigmented (C57BL/6J, n = 10) and albino (BALB/cJ, n = 3) mice were imaged in vivo. Enucleated eyes were processed for light and electron microscopy. Using well-accepted reference surfaces, we compared micrometer-scale axial reflectivity of visible-light OCT with subcellular organization, as revealed by 9449 annotated EM organelles and features across four pigmented eyes. Results In study 1, outer nuclear layer reflectivity peaks coincided with valleys in heterochromatin clump density (−0.34 ± 2.27 µm limits of agreement [LoA]). In study 2, band 2 depth on OCT and IS/OS junction depth on EM agreed (−0.57 ± 0.76 µm LoA), with both having similar distributions. In study 3, RPE electron dense organelle distribution did not agree with reflectivity in C57BL/6J mice, with OCT measures of RPE thickness exceeding those of EM (2.09 ± 0.89 µm LoA). Finally, RPE thickness increased with age in pigmented mice (slope = 0.056 µm/mo; P = 6.8 × 10 −7 ). Conclusions Visible-light OCT bands arise from subcellular organization, enabling new measurements in mice. Quantitative OCT–EM comparisons may be confounded by hydration level, particularly in the OS and RPE. Caution is warranted in generalizing results to other species.

Multiple Bioenergy-Linked OCT Biomarkers Suggest Greater-Than-Normal Rod Mitochondria Activity Early in Experimental Alzheimer's Disease

“…A consensus clinical lexicon, based on a body of supporting literature, suggests that band 2 measured with spectral-domain OCT (SD-OCT) represents the inner segment EZ, whose volume is normally 75% filled with mitochondria. 30 – 35 Our functional SD-OCT studies strongly support this consensus interpretation that mitochondria are the major contributor to band 2. 36 , 37 For example, we have shown that the EZ reflectivity profile shape measured with SD-OCT agrees with mitochondria redistribution within the inner segment ellipsoid measured from electron microscopy in mice with different mitochondria activity modulated by either strain or light level.…”

High-Resolution Optical Coherence Tomography in Healthy Individuals Provides Resolution at the Cellular and Subcellular Levels

“…Similar results were obtained in other studies on photoreceptor nuclei using different combined OCT methods and histological comparisons. 10 , 25 – 27 Owing to the improved resolution and lower speckle noise of High-Res OCT, dark spots within hyporeflective bands can be discovered more frequently and clearly compared with Spectralis HRA-OCT.…”

“…So far, only a few studies show visualization of cellular features by using enhanced technology such as visible light OCT or adaptive optics OCT. 10 , 11 However, both systems have their limitations and challenges when it comes to implementation in clinical practice.…”

Ocular Pigmentation Impact on Retinal Versus Choroidal Optical Coherence Tomography Imaging in Preterm Infants

“… 22 , 23 Melanin in these two pigmented layers reflects or absorbs the near-infrared light used in OCT imaging, potentially limiting penetration of the signal to deeper structures. 24 – 26 One previous study of 86 preterm infants assessed the impact of maternal identification of race on OCT imaging of the choroid but did not investigate fundus pigmentation as a relevant biological variable. 27 They found that maternal self-identification as African American race was associated with decreased chorio–scleral junction (CSJ) visibility among term-age preterm infants.…”