In vivo cross-sectional retinal anatomy and pathology as visualized by OCT has revolutionized the study of retinal disease. Since its original commercial deployment, OCT technology has become much faster, has an improved axial resolution, and has incorporated frame-averaging software capable of increasing the signal-to-noise ratio. Although these advances have allowed additional layers to be resolved, particularly in the outer retina, they do not routinely visualize HFL as distinct from the ONL. However, the optical contrast necessary to visualize this layer can be brought out by altering the angle of the OCT entry beam on HFL, which consequently alters its reflectivity relative to the ONL. Thus, in addition to the technology used to generate a scan, the technique in which the OCT data are collected can directly determine what is visualized. Future software could allow registration of images acquired at different pupil entry positions to provide the clearest overall delineation of HFL from the ONL across an entire B-scan.
We observed that as the SD-OCT entrance beam moved closer to the edge of a dilated pupil, the reflectivity of HFL on that side of the fovea was reduced whereas HFL reflectivity on the opposite side of the fovea increased. Because the intensity of reflected light varies with pupil entry position, HFL exhibits directional reflectance. We confirmed this finding was a consistent effect using a variety of scan protocols, multiple subjects, and two separate commercially available SD-OCT systems. We found this effect to be accentuated by, but not dependent on, the use of frame averaging to reduce speckle noise.
Accompanying this directional reflectance is the apparent tilt of the B-scan. This is “apparent” in that the real geometry of the eye does not change when using an eccentric entry position of the SD-OCT beam, but the optical path lengths of the scan do change. We found that the pupil entry position by which the SD-OCT B-scan appeared flat varied between subjects and did not necessarily correspond the geometric center of the pupil. However, once that position was identified, there was a predictable effect achieved by movement of the OCT entry beam. As the SD-OCT beam is moved nasally, the distance light must travel to and from the nasal macula is shorter than the path it must take to and from the temporal retina. The resultant B-scan is consequently tilted in appearance. The degree of this tilt served as an indirect means to calculate the entry beam position relative to the scan direction and was directly related to the relative reflectivity of HFL when normalized by the IPL in all subjects analyzed. The IPL was found to be relatively invariant in reflectivity at each of the eccentricities and, accordingly, served as a control for the directionally reflective HFL. Although HFL intensity varied without this normalization, normalization isolated the effects of directional reflectance from the overall image intensity variation induced by other pupil position–dependent changes, such as variable ocular transmission and scatter.
Directional reflectivity is a property shared by several retinal structures in which the optical principles behind their occurrences are well understood. For example, in photoreceptor IS and OS, the optical Stiles-Crawford effect22
exists because of photoreceptors acting as waveguides, each directed at the center of the pupil. Additionally, the surface reflection visualized from the ILM in young eyes is caused by specular reflection from the smooth interface between two media of different refractive indices.
The directional reflectivity of the retinal nerve fiber layer (RNFL) has been well characterized by in vitro experiments.23,24
The conclusion of these experiments was that a ray of light incident on the RNFL scatters into a conical sheet coaxial to the fiber bundle with the same angle relative to the fibers as the incident ray. Based on this experimental evidence, reflectivity of the human RNFL was modeled as light scattering by cylinders, and the implications of variability caused by directional reflectance on clinical measurements was discussed.25
The mechanism of the directional reflectivity displayed by HFL was most consistent with that of the RNFL because both these structures are composed of long cylindrical axons. The magnitude of the change in HFL reflectivity we observed because of the change in beam position was also consistent with that observed for the RNFL in rat retina.24
However, the RNFL appeared highly reflective when imaged through the center of the pupil until it turned to enter the optic canal, whereas HFL did not. This difference can be explained by the oblique orientation of HFL, which was due to axons running from photoreceptor nuclei toward the horizontally displaced cells of the inner retina. As the beam was displaced eccentrically in the pupil, the angle at which these light rays were incident on HFL changed, and consequently, so did their primary scattering angle. As the angle of incidence approached normal to HFL, increasingly more light was scattered back from this layer to escape the pupil and be visualized as OCT hyperreflectivity. Alternatively, with shallower angles of incidence, light scattered further away from the exit pupil, creating a hyporeflective HFL on SD-OCT ().
Figure 7. Unscaled schematic of HFL reflectivity through an eccentric pupil. Light rays from the edges of a B-scan centered on the fovea from an eccentric entry position are shown. These light rays encounter the obliquely oriented HFL (white lines within thickened (more ...)
Quantitative estimates suggest results that are consistent with our observations. Using the Bennett-Rabbetts' model eye,26
the approximate angle of incidence of light rays emanating from an entry position 3 mm from the anatomic axis was calculated to be approximately 8.5° to the retina. The angle of HFL in was calculated to be approximately 8°. These estimates predict that on the side of the fovea opposite the pupil entry position, light would be incident on HFL nearly normal to the axon orientation, resulting in maximal reflectivity back toward the exit pupil. This was consistent with the data presented in , showing maximal intensity of HFL occurred near 8°, and declined beyond that point nasally. Again consistent with our data, this model predicts that light rays would encounter HFL on the same side of the fovea as the entry beam at an angle of incidence around 17°, resulting in a reflection directed away from the exit pupil and appearing hyporeflective on OCT.
Recognition of the optical principles governing HFL reflectivity provides an explanation for the “unexpected” reflectivity in this layer because of pathology that affects the retinal geometry. The images in and demonstrate reflectivity changes caused by alterations in the normal geometry of the retina, where pigment epithelial detachment, drusen, or subretinal fluid alters the angle of the cone of light reflecting from HFL. Optical changes in HFL are introduced by these protuberances by elevating and changing the orientation of its fibers relative to the pupil such that different segments of HFL may appear to be hyporeflective and hyperreflective overlying a single deformation. Diffuse hyperreflectivity accompanying drusen has been commented on previously and has been theorized to represent a “degenerative cellular process.”19
Although this explanation is possible, the anatomic location of hyperreflectivity attributed to drusen in corresponds to HFL, and its intensity can be seen to vary substantially with pupil entry position, supporting the idea of an optical effect. Now that a means to visualize HFL has been recognized, similar types of pathology could be dynamically imaged to further distinguish optical alterations from independent pathologic processes.
Identification of the true dimensions of the ONL is critical to clinical studies that aim to accurately measure macular photoreceptor nuclei thickness without the confounding effect of HFL. Recognition of a means by which to optically section the previously homogeneously reflecting tissue located between the ELM and OPL affords this distinction. It is possible that the nuclear thinning reported by Schuman et al.19
might have been an underestimate and that the segmentation of ONL and HFL independently would have further bolstered the importance of their measurements. For OCT data that have already been acquired, the ability to infer true photoreceptor thickness from histologic data will be useful but is limited. The heterogeneity evident in demonstrates that HFL thickness varies between persons and by eccentricity from the foveal center even in subjects with normal vision. This variability is expected given observed variation in foveal cone density27
and foveal pit morphology.28
This study did not seek to convey average or normal values of the HFL and ONL thicknesses. Accurate reporting of normative data would depend on a much larger number of subjects and on several parameters that were not precisely measured in this study, including axial length and refractive error. Furthermore, it is unclear whether HFL intensity and thickness measurements 1 mm away from the foveal center are more important than measurements at other eccentricities. This location was selected as a convenient distance from which to measure because of the substantial contribution of HFL that was not confounded by vascular shadowing. Additionally, the choice of rectangle size used to measure the intensity of reflectivity was not necessarily optimal. However, the area was large enough to generate a reproducible distribution of values with a small SD, yet it was small enough to reliably fit within HFL and the IPL and was invariant to shifts of several pixels in any direction. Future studies can examine volumetric data in larger numbers of patients along all A-scans at eccentric entry positions to determine normative values of true ONL thickness and intensity visualized by SD-OCT.
The striking image quality achievable with SD-OCT systems makes it tempting to directly correlate retinal layers visualized with SD-OCT images with retinal histology. However, these are not identical; they vary based on the optical properties of retinal tissue. Importantly, an SD-OCT image depends not only on the particular system used but on the technique by which an image is acquired. Revealing the directional reflectivity of HFL by altering the beam entry position is an example of this phenomenon and can be exploited to gain a more thorough understanding of the retina in health and disease.