Imaging and quantifying ganglion cells and other transparent neurons in the living human retina

Abstract

Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging-using predominately singly scattered light-to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: (i) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; (ii) performing 3D subcellular image registration to avoid motion blur; and (iii) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease.

Keywords: adaptive optics; optical coherence tomography; organelle motility; retina; retinal ganglion cells.

Fig. 1.

Averaging registered AO-OCT images improves clarity of GCL somas. Magnified view of the same small patch of retina is shown with different amounts of averaging (n = 1, 30, and 137 images). Images are from 12–13.5° temporal to the fovea in subject S3. Plot shows the contrast-to-noise ratio (CNR) of 120 individual GCL somas computed as a function of images averaged (Materials and Methods). Error bars denote ±1 SD. CNR increase follows the square root of the number of images (dashed curve).

Fig. 2.

Cellular structures of the inner layers of the retina using AO-OCT. (A) Yellow square at 12–13.5° temporal to the fovea in subject S3 denotes location imaged with AO-OCT. (B) Three-dimensional perspective of registered and averaged AO-OCT volume with green dashed line denoting cross-section of inner retina shown in C. Yellow arrow indicates same GCL soma in C and F. Images shown in D–G were extracted at depths of 0, 13, 22, and 46 μm below ILM. Scale bar in G also applies to D–F. (D) Surface of ILM. Bright, irregular star-like structures sparsely cover the surface of the ILM and are consistent in appearance with individual astrocyte or microglial cells. (E) A complex web of nerve fiber bundles of varying size disperse across the NFL. Some have a diameter as large as 30 μm (blue arrow), which compares to our previous AO-OCT observations (48). Others are as small as 3 μm, which matches the caliper of a single large GC axon. An arteriole/venule branches on the left. GCL somas appear between the overlying bundles near the image bottom (green arrow). (F) A mosaic of GCL somas of varying size tile the layer. Red arrow points to a large soma. Caliper of arteriole/venule in E is sufficiently large that it extends into the GCL. Note the distinct edges of the vessel walls (blue and white arrows) and the tight abutment of GCL somas. (G) The dense synaptic connections between axons of bipolar cells and dendrites of ganglion and amacrine cells present as a uniform mesh of high spatial frequency irregularities in the IPL. COST, cone outer segment tip; IS/OS, inner segment/outer segment junction; ONL, outer nuclear layer; OPL, outer plexiform layer (Movie S1).

Fig. 3.

En face images extracted from GCL at increasing retinal eccentricity of subject S4. A mosaic of GCL somas is observed at each eccentricity. (Bottom Right) GC soma density is plotted along the horizontal meridian of the macula. Retinal eccentricity is converted to millimeters to compare with histology data (10). AO-OCT temporal data are the average from four subjects and nasal is from S4. Error bars denote ±1 SD.

Fig. 4.

Properties of soma size, reflectance, and pooling of cone signals. (A) Representative GCL soma size distribution (S4) is color coded by retinal eccentricity. (B) Average GC soma diameter obtained by Gaussian fits to the four subjects in temporal retina with measurements reported in the literature for humans (5, 10, 12, 26, 29, 34). Error bars denote ±1 SD unless labeled with an r to denote minimum-to-maximum range. M and P denote midget and parasol cells. Labels along x axis report retinal location of measurement. fm, foveal margin; pm, papillomacular; pr, peripheral retina. (C) Representative reflectance of 637 GCL somas at 12–13.5° temporal to the fovea in subject S4. Blue line shows linear regression curve. (D) mRGC-to-cone ratio for four subjects is plotted with Watson’s histology-based model for humans (figure 14 of ref. 21) and fovea ratios in table 1 of Drasdo et al. (20).

Fig. 5.

Cells at different depths in the same retinal patch of subject S4 as visualized with AO-OCT. (A) Three-dimensional perspective of registered and averaged AO-OCT volume with colored lines denoting retinal depths at which the en face images in B–F were extracted. Images depicting individual NF bundles (B), GCL somas (19,162 cells per mm²) (C), suggestive somas of bipolar (green arrow) and displaced GCs (white arrow) near the IPL interface (D), cone photoreceptors (16,341 cells per mm²) (E), and RPE cells (4,893 cells per mm²) (F). Black arrows in C–F indicate the same blood vessel and its shadow. The en face images were extracted from volumes acquired at 3–4.5° retinal eccentricity with system focus shifted axially to maximize sharpness of the cell layer of interest.