Imaging individual neurons in the retinal ganglion cell layer of the living eye

Abstract

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Keywords: adaptive optics; imaging; photoreceptors; retina; retinal ganglion cells.

Fig. S1.

On-axis diagram of the various detection schemes used in AOSLO. In confocal (A), a small (e.g., 2 ADD) pinhole passes the light from core of the PSF. In offset-aperture (B), a larger aperture (e.g., 10 ADD) is offset (e.g., by 7 ADD) from the center of the PSF. In dark-field (C), a narrow (e.g., 2 ADD) filament (black rectangle) blocks the central core of the PSF and light is collected through a larger aperture (e.g., 10 ADD). In split-detection (D) the central core of the PSF is directed to a separate confocal channel using an annular mirror with a small (e.g., 2 ADD) reflective portion (gray circle), the light from each half of the remaining larger (e.g., 10 ADD) area is transmitted and then split with a knife edge and directed into two separate detectors. In the radial multioffset detection pattern (E), an aperture is sequentially positioned at a fixed distance (e.g., 6, 11, or 16 ADD) from the PSF at several different angles (e.g., every 45°). In a triangular multioffset detection pattern (F), the aperture is positioned at several points arranged in a triangular grid. Black outlines enclose detection areas. All images are drawn to scale with respect to the size of a theoretical Airy disk (red circle). It should be noted that this comparison is of the detection scheme only and ignores any optional illumination manipulations [e.g., in some forms of dark-field (22)] or digital manipulations after image formation [e.g., in split-detection (24) and multioffset].

Fig. 1.

Offset-aperture images (A–K and M–W) show characteristic intensity gradients across cones. Images positioned to reflect sampling pattern (aperture: ∼4 ADD; offset: ∼8–21 ADD). SD of offset-aperture images (X) reflects asymmetry as cones appear as rings. Differencing enhances contrast; Y is the difference of P and H. Confocal (L) and TPEF (Z) shown for comparison. Light was focused to maximum cone TPEF, assumed to be at plane of cone outer segments. Images are 100 × 100 µm.

Fig. S2.

Zoomed-in views of TPEF (A), confocal (B), and multioffset (C) images from Fig. 1 are shown here to illustrate that every cone was visible in all imaging modalities but rod visibility varied. Field-of-view of A–C and E–H is 40 × 40 µm; square in D denotes location of zoomed-in views. Manually marked cone and rod locations are shown in E–G and are overlaid in H to show positional variability. In H, yellow denotes overlap of TPEF and confocal; magenta denotes overlap of TPEF and multioffset; cyan denotes overlap of confocal and multioffset and white denotes overlap of all three.

Fig. S3.

Offset-aperture images (A–D and F–I) can be combined in numerous ways into multioffset images (J–N) that can appear similar to split-detection images (O) and reveal cones in AMD (N, arrow) that were not visible in confocal images (E, arrow). Offset-aperture images obtained with visible light (680 nm) using a radial sampling pattern (∼9 ADD aperture; ∼6 ADD offset; 45° intervals) in an area of the retina that appears normal on clinical examination in a patient with AMD (A–D and F–I); Inset denotes aperture position with respect to center of PSF (denoted with 1 ADD gray dot). Confocal image from NIR channel is shown in E. Multioffset images (J–N) can be generated in many ways, including: (i) difference of two aperture positions (J, K, N); (ii) difference of averages of two aperture positions (L) and difference of average of B and C and G and H; and (iii) difference of averages of three positions (M) and difference of average of A, D, G and C, F, I. Aperture positions differenced denoted in Insets of J–N. The multioffset image with the best subjective contrast was often but not always (N) obtained from aperture positions on exact opposite sides of the PSF. (Scale bars, 50 µm; scale bar in E applies to A–N.)

Fig. 2.

Cones were not visible in some areas adjacent to drusen or on margin of hypo-autofluorescence (Fig. S4) in confocal AOSLO (A, arrows) but multioffset images (B, arrows) showed a contiguous cone mosaic in some of these regions. Drusen substructures were seen in multioffset images as well circumscribed circular features (B, arrowhead); cones were visible at corresponding locations in confocal image (A, arrowhead). Corresponding clinical images are shown in Fig. S4. (Scale bar, 50 µm.)

Fig. S4.

Color fundus photography (A and C) and autofluorescence SLO (B and D) reveal gross drusen structure and patches of hypo-autofluorescence, suggestive of early atrophy in AMD. Squares in A and B reflect field-of-view of images below; squares in C and D reflect field-of-view of AOSLO images shown in Fig. 2; arrows and arrowheads are at their corresponding locations from Fig. 2. (Scale bars, 500 µm in A and 200 µm in C.)

Fig. 3.

Confocal (Left column: A, D, G, J, M, P), TPEF (Center column: B, E, H, K, N, Q), and multioffset (Right column: C, F, I, L, O, R) AOSLO images at several focal planes in a monkey. Cell somas were visible in the ganglion cell layer in TPEF (B) and multioffset (C) images but not confocal (A). Focal planes between the outer and inner layers contained laminar and mosaic-like structures in TPEF (E and H) and multioffset (F and I). Photoreceptor somas may be visible in TPEF and multioffset at the next focal plane (K and L). When focused at a more inner plane, cones appeared differently in multioffset (O) than at a deeper plane (R), where they appear similar to cones in split-detection. Approximate axial depth relative to the ganglion cell layer: (A–C) 0 µm; (D–F) 36 µm; (G–I) 69 µm; (J–L) 102 µm; (M–O) 140 μm; (P–R) 182 µm. Retinal eccentricity is ∼4 mm from the fovea along the horizontal meridian within the temporal raphe. (Scale bar, 25 µm.)

Fig. S5.

The TPEF image shown in Fig. 3B is shown here inverted and smoothed with a three-pixel-wide Gaussian blurring filter here in A. The segmentation image from the multioffset image from Fig. 3C is shown here in B. These images are merged in C to show that the dark regions of the TPEF image (shown as green regions in C) often reside within the somas visible in the multioffset images. Images are 175 × 175 µm.

Fig. S6.

Blood vessel contrast can be enhanced or nearly completely minimized depending on multioffset configuration. The contrast of the blood vessel running diagonally in A from lower left to upper right is enhanced with this multioffset configuration (A, Inset) but minimized in B with the orthogonal configuration (B, Inset). Images obtained from a normal human retina. (Scale bar, 50 µm.)

Fig. 4.

Somas were not visible in offset-aperture images nearest to the PSF (A) but were visible at more distant positions (B and C). The SD image increased the contrast of some somas, particularly cells adjacent to a blood vessel on the left edge (D). Multioffset difference images enhanced somal contrast (E and F), but not all cells were visible across each image (e.g., cell in E, arrowheads vs. cell in F, arrows). Averaging many difference images and enhancing local contrast with contrast limited histogram equalization, improved cell contrast, and visualized cells across the entire field-of-view (G). Some cells had visible subcellular structures (H–K). Arrows in G denote locations of H–K. Somas outlined with solid lines (I and K) contained putative nuclei (dashed circles) and nucleoli (dotted circles). Insets in A–G show detection pattern; shaded circle is aperture position of offset-aperture image (A–C) or multioffset images (D–G); dot is 1 ADD and denotes PSF center; aperture diameter: ∼4 ADD; offsets centered from ∼8–21 ADD. Image from raphe of macaque (∼4 mm from fovea). (Scale bar in E, 25 µm and applies to A–G; H–K are 50 µm × 50 µm.)

Fig. S7.

Multioffset images of ganglion cell layer neurons in monkeys with high light levels (∼7 mW) required for TPEF (A) are still visible at the lower light levels (∼270 µW) typically used for human imaging with the same size aperture (B) or with an aperture with twice the diameter (C). (Scale bar, 25 µm.)

Fig. 5.

Multioffset images of ganglion cell layer neurons in the temporal retina (∼3.5 mm from fovea) along the raphe (A) and superior to the raphe (∼3.5 mm temporal, ∼1.5 mm superior of fovea) (B) from two different human eyes. (Scale bar, 50 µm.)

Fig. S8.

Corresponding confocal images (A and B) from the locations of the multioffset images shown in Fig. 5 show no discernable cell somas. Corresponding segmentation images (C and D) show the locations of the cell somas identified from the images shown in Fig. 5. (Scale bar, 50 µm.)

Fig. S9.

Confocal (A), multioffset (B), and segmentation images (C) from an area superior to the raphe in a monkey shows characteristic pattern of ganglion cell layer somas lining up along the axon bundles of the nerve fiber layer. Multioffset image is a difference of two offset-aperture images. (Scale bar, 50 µm.)

Fig. 6.

Somal diameter distribution (A) was within the expected range (B). Means (±2 SD) are compared with values from the literature in B. Data points in B are mean (±2 SD) of measurements digitized for eccentricities of 2–4 mm from figures 4 in ref. , 8 in ref. , and 16 in ref. . Values are from text of ref. for a 10-mm-diameter area centered on the fovea and from a retinal eccentricity of 4–8 mm for ref. .

Fig. S10.

Histogram shows distribution of somal area for the same data as shown in Fig. 6.