Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

Abstract

The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°-15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders.

Keywords: (110.1080) Active or adaptive optics; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology; (330.5310) Vision; photoreceptors.

Fig. 1

Reflectance images of the human photoreceptor mosaic at three retinal locations along the temporal meridian for subject DLAB_0008, collected using 680 nm light and 0.4 Airy disk pinhole size. The same images are shown with linear (top row) and logarithmic (bottom row) grayscales, to facilitate visualization of the rod mosaic. The scale bars are 10 μm across.

Fig. 2

The two images on the left and middle were collected in an excised primate retina, imaged in a bright field microscope in transmission [45]. These images, reproduced with permission from the Journal of Neuroscience, show the cone and rod outer segment tips, respectively, as bright spots. The image on the right shows an in vivo image from WLAB001 at similar eccentricity, collected using 775 nm light and 0.6 Airy disk pinhole size. The scale bar is 5 μm across.

Fig. 3

Reflectance images of the human photoreceptor mosaic at 10° temporal to the fovea for subject JC_0138, collected using 680 nm light and 1.1 Airy disk pinhole size. From top to bottom the images are: a single frame, a registered average of 50 frames, and registered average of 6 batches of 50 frames, collected over a 6 hour period, 1 hour apart. The scale bars are 20 μm across.

Fig. 4

Comparison of in vivo rod and cone metrics with those from Curcio et al. [1]. Shown on the left is a plot of the ratio of rods to cones as a function of retinal eccentricity. The solid line is the mean of Curcio’s measurements taken in the temporal meridian, and filled circles correspond to the data from this study. On the right is a plot of photoreceptor density as a function of retinal eccentricity. Density estimates for our subjects for rods and cones are shown as open squares and open circles, respectively. Also plotted is the mean rod (solid line) and cone (dashed line) density values reported by Curcio et al. [1] for the temporal meridian.

Fig. 5

Analysis of the regularity of the peripheral photoreceptor mosaic. Shown in a is the 6-hour averaged image (logarithmic display) from subject JC_0138, taken at about 10° temporal to fixation, collected using 680 nm light and 1.1 Airy disk pinhole size. Color-coded Voronoi domains associated with each cell are shown in panel b), where the color indicates the number of sides on each Voronoi polygon (magenta = 4, cyan = 5, green = 6, yellow = 7, red = 8, dark blue = 9). Regions of six-sided polygons indicate a regular triangular lattice, while other color mark points of disruption of the mosaic. Panel c shows the color-coded Voronoi domains associated with just the cone photoreceptors in the image.

Fig. 6

Reflectance image of the human photoreceptor mosaic from subject JC_0138, collected using 680 nm light and 1.1 Airy disk pinhole size and displayed with linear (left) and logarithmic (right) gray scale mappings. The image is a montage of two overlapping locations, stitched together using i2k Retina (Dual Align, LLC, Clifton Park, NY, USA). The arrows point to some of the rod photoreceptors closest to the foveal center (significantly smaller than surrounding cones), which is located at the bottom right corner. The scale bars are 50 μm across.

Fig. 7

Reflectance images of the human photoreceptor mosaic from subject DLAB_007 at 10° temporal along the horizontal meridian at different retinal depths, shown with linear (top row) and logarithmic (bottom row) gray scales. The image series was collected by using 680 nm light and a 0.4 Airy disk pinhole size. The zero depth point indicates the innermost image of the stack, and, thus, increasing values indicate foci closer to the retinal pigment epithelium (RPE). The scale bars are 10 μm across.

Fig. 8

Axial intensity profiles for the cone and rod photoreceptor mosaics shown in Fig. 7. The (very small) error bars correspond to the errors associated with identifying the boundary of the cones in the images

Fig. 9

Reflectance images of the human photoreceptor mosaic collected using 680 and 775 nm light and 1.1 and 1.6 Airy disk pinhole sizes, respectively, shown with linear (left) and logarithmic (right) gray scales. Scale bars are 10 μm across.