High-Resolution Imaging of the Retinal Nerve Fiber Layer in Normal Eyes Using Adaptive Optics Scanning Laser Ophthalmoscopy

To conduct high-resolution imaging of the retinal nerve fiber layer (RNFL) in normal eyes using adaptive optics scanning laser ophthalmoscopy (AO-SLO).AO-SLO images were obtained in 20 normal eyes at multiple locations in the posterior polar area and a circular path with a 3-4-mm diameter around the optic disc. For each eye, images focused on the RNFL were recorded and a montage of AO-SLO images was created.AO-SLO images for all eyes showed many hyperreflective bundles in the RNFL. Hyperreflective bundles above or below the fovea were seen in an arch from the temporal periphery on either side of a horizontal dividing line to the optic disc. The dark lines among the hyperreflective bundles were narrower around the optic disc compared with those in the temporal raphe. The hyperreflective bundles corresponded with the direction of the striations on SLO red-free images. The resolution and contrast of the bundles were much higher in AO-SLO images than in red-free fundus photography or SLO red-free images. The mean hyperreflective bundle width around the optic disc had a double-humped shape; the bundles at the temporal and nasal sides of the optic disc were narrower than those above and below the optic disc (P<0.001). RNFL thickness obtained by optical coherence tomography correlated with the hyperreflective bundle widths on AO-SLO (P<0.001)AO-SLO revealed hyperreflective bundles and dark lines in the RNFL, believed to be retinal nerve fiber bundles and Müller cell septa. The widths of the nerve fiber bundles appear to be proportional to the RNFL thickness at equivalent distances from the optic disc

High-magnification RNFL images using AO-SLO of the area indicated by white boxes in Fig. 2

<p>(<b>a</b>–<b>e</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033158#pone-0033158-g002" target="_blank">Fig. 2</a> corresponds to <b>A</b>–<b>E</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033158#pone-0033158-g005" target="_blank">Fig. 5</a>). <b>A</b>, Image near the optic disc indicated by a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033158#pone-0033158-g002" target="_blank">Fig. 2</a>. <b>B</b>, Image 2 mm above the fovea. <b>C</b>, <b>D</b> Images at the temporal raphe. Bridges can be seen among the hyperreflective bundles (<i>arrows</i>). <b>E</b>, Image of the area around the papillomacular bundle. The dark lines among the hyperreflective bundles are narrower around the optic disc (<b>A</b>) than at the temporal raphe (<b>C</b>, <b>D</b>). The hyperreflective bundles on the nasal side of the fovea (<b>E</b>) are thinner than those above (<b>B</b>) or below the fovea. Scale bar = 100 µm (A–E).</p

Optical system diagram.

<p>The adaptive optics scanning laser ophthalmoscopy (AO-SLO) system comprises 4 primary optical subsystems: the AO subsystem including the wavefront sensor, the high-resolution confocal SLO imaging subsystem, the wide-field imaging subsystem, and the pupil observation subsystem for initial alignment of the subject's pupil with the optical axis of the AO-SLO system by adjusting the chin rest. The AO subsystem incorporates a liquid-crystal spatial light modulator (LC-SLM) based on liquid crystal-on-silicon (LCOS) technology. The LC-SLM and wavefront sensor are controlled using a custom software to reduce the wavefront errors. The results of high-resolution imaging are linked to the results of the wide-field imaging subsystem, which are obtained by the line-scan SLO system.</p

Measurement of the width of hyper-reflective bundles.

<p>To measure the width of individual hyper-reflective bundles, 3–8 bundles were randomly chosen from 1 AO-SLO image (1.5°×1.5°). The digital caliper tool was used to measure the width at a minimum of 3 points in 1 bundle by 2 independent experienced graders (i.e., the bundle width of this area was defined as the mean width of 18 points).</p

Comparison of the number of peaks of plot profile between AO-on images and AO-off images.

<p><b>A</b>, <b>B</b>. AO-on image focused on the RNFL. Mean gray value of images was plotted (190 pixel width) along a line that vertically crosses the hyper-reflective bundles. <b>C</b>. Plot profile of gray value along the yellow line of <b>B</b>. The number of peaks, which was defined as more than 20 gray values compared to the neighboring baseline, was 8. <b>D</b>, <b>E</b>. AO-off image of same area. <b>F</b>. Plot profile of gray value along the yellow line of <b>E</b>. The number of peaks was zero.</p