Figure 3.

Locations of significant light-dependent changes in apparent diffusion coefficient (ADC) are shown relative to known outer retinal morphology. Unique light-dependent changes were found for ADCs measured parallel to the optic nerve (ADC║), perpendicular to the optic nerve (ADC┴), and in diffusion anisotropy (i.e., ADC┴ - ADC║). Central retinal rods and associated structures are depicted to scale, based on the well documented layered microstructure of the rat retina, with rod nuclei (N) and rod inner and outer segments (RIS, ROS) anterior to the retinal pigment epithelium (RPE). The rod cytoplasm is shown in white, interstitial space (and rod nuclei) in light gray, and processes from Müller glia in black between rod somas. The dimensions of all structures are illustrated accurately based on the extensive body of literature for the rat retina, including the diameters of rod nuclei [43,53], the connections between rod nuclei and other structures (“rod fiber” in [43]), and the connection between RIS and ROS [43,54]. Note that the dimensions of each cellular element are based on an average of at least two literature sources. For illustrative purposes, the main panel (which shows the ║ direction oriented from left to right) displays all structures centered on same two-dimensional plane. Though this is a fair depiction of RIS and ROS, which are spaced in a regular hexagonal lattice [32,55], the arrangement of rod somas is less orderly, and it is generally not possible to see several neighboring rod nuclei at full diameter in a single histological section. The gray within ROSs is a depiction of the several hundred photopigment-laden disks (approximately 30.5 per μm of ROS length [54-60]) at low magnification, which is clarified in the inserts. Results and landmarks are depicted against the %thick scale used for the mfADC data. Optical coherence tomography images of the rat retina [61-65] demonstrate that the distance from vitreoretinal border to the choroid is approximately 200 μm, with the following divisions: the border between the outer plexiform layer and outer nuclear layer (i.e., the anterior-most position of the photoreceptor nuclei) occurs approximately 101 μm from the vitreoretinal border (i.e., 50%thick); the division between outer nuclear layer and the bacillary layer (posterior outer retina, containing rod inner and outer segments) occurs at approximately 143 μm (72%thick); and the posterior limit of the rod outer segments occurs at approximately 187 μm (94%thick) from the vitreoretinal border, with the remaining space to the choroid (at 100%thick) occupied by RPE. Histological studies of the rat retina are in good agreement with those figures, showing that rod outer segments are approximately 27 μm long, while rod inner segments are approximately two-thirds that length [32,43,53,55,60,66], for a combined approximately 45 μm span for the posterior half of the outer retina. The outer nuclear layer (which we refer to as the anterior outer retina) is approximately 39 μm thick [58-60,66-69], and the distance between the outer nuclear layer and vitreoretinal border is approximately 106 μm [67-69]. Including an approximately 8 μm RPE [68], histological studies therefore describe the anterior outer retina spanning from 53% to 73%thick, and a posterior outer retina from 73% to 96%thick. Left insert (║ into/out of plane): Cross sections show the radii of ROS (r_ROS) and disks contained within (r_Disk), along with the thin rim of cytoplasm that runs the length of the ROS between the disk and ROS membranes. The depiction of ROS and disk radii are based on published electron micrographs, where r_ROS is approximately 0.73 μm [32,43,54,55,57,58] and the difference between r_ROS and r_Disk is approximately 0.03 μm [54-56,60]. The inter-ROS spacing shown here is based on known photoreceptor density: an approximately 10 µm² area is illustrated here, and the literature’s ranges for packing density range from 3.1 to 4.0 rods per 10 µm² [43,59,70]. Right insert (║ left/right): Fine detail of the ROS shows the location of intra-disk (d) cytoplasmic (cy) and interphotoreceptor (i) water between membranes (black lines). The total disk thickness is depicted as equal to the thickness of the between-disk cytoplasmic space, based on available information available for the rat [54-56,60], and the pattern found in several other species [71]. The range of literature values may be due to the sensitivity of ROS disks to fixation method [54,72].