Optical coherence tomography (OCT) uses infrared light to interferometrically derive optical reflectivity information varying by depth in living tissues.⁷ The use of broadband light sources, a spectrometer, and the application of signal processing techniques has culminated in commercial spectral domain (SD) OCT systems that are capable of imaging with a 5-μm axial resolution in retinal tissue.^8,9 These improvements, along with acquisition speeds fast enough to permit frame averaging without significant motion artifact, contribute to improved image quality in SD-OCT devices capable of identifying structures that could not previously be resolved.¹⁰

Despite advances in SD-OCT hardware and software, HFL visualization has remained elusive. A seminal paper published in 2004 comparing in vitro OCT images of monkey fovea to histology recognized HFL as a major layer of the retina that could be visualized in vitro and that should be accounted for in vivo.¹¹ However, since that publication, HFL has not been included in diagrams of retinal layers visualized by OCT, likely because of the inability to distinguish a change in reflectivity at the interface between the HFL and the outer nuclear layer (ONL).^12–14 Furthermore, segmentation algorithms, normative thickness data, and ONL measurements overlying drusen have recently been published that do not recognize the contribution to macular thickness provided by HFL to the ONL.^15–19 Although the need to account for the presence of HFL in OCT images has recently been recognized, the proposed means to measure the contribution of the HFL was an inferential normative model based on high-quality pathologic specimens rather than by a direct means of visualization (Curcio CA, et al. IOVS. 2010;51:ARVO E-Abstract 2286). The only explicit mention of the optical conditions in which HFL may be visualized with OCT can be found in a letter to the editor and was not experimentally validated.²⁰ In our study we describe a systematic method that can be applied to commercial SD-OCT systems to directly visualize and quantify HFL. Additionally, we provide an optical explanation of this phenomenon and demonstrate its clinical relevance.

The position of the entrance beam could be determined by the pupil camera on Cirrus scans. As the beam entry position was moved, there was a corresponding change in the apparent tilt of the B-scan. In several cases, the geometric center of the pupil did not correspond to the entry position, yielding a flat B-scan. Changes in beam entry position in the direction of the scan could also be determined directly by monitoring the tilt of the B-scan relative to the axially oriented direction of A-scans, most notably by the hyporeflectivity deep to retinal blood vessels. This was found to be a more precise means of determining pupil entry position than relying on the pupil camera, where image distortions increased the likelihood of registration errors.

Movement toward the nasal pupil resulted in a tilt of the B-scan such that the macula temporal to the fovea appeared lower in the scan than the macula nasal to the fovea. Concurrently, the intensity of pixels corresponding to the temporal HFL increased, whereas the nasal HFL decreased in intensity. The pixel values corresponding to HFL were normalized by the IPL because the intensity from HFL could be altered not only by the intrinsic reflectivity of the tissue but also by variations of incident intensity of the scan given the variable ocular transmission and scatter. The IPL intensity values were found to have a mean coefficient of variation of 6% across subjects. Given that SD-OCT images are rendered on a logarithmic scale, this was extremely invariant compared with HFL, where the mean coefficient of variation was 27% across subjects. Normalization by the IPL resulted in a direct relationship between scan angle and HFL intensity and demonstrated its directional reflectance properties (Fig. 3). Reflectivity of the nasal macula increased as scan angle decreased, which corresponded to temporal displacement. In two patients there was a fall-off of relative intensity of the nasal HFL after 9°. Conversely, as scan angle increased, reflectivity of the temporal macula increased. There was no resultant fall-off for the temporal retina, with maximum relative intensity obtained at 6°.

Measurements of HFL thickness as a percentage of the total thickness between the ELM and the OPL was performed when HFL intensity was in the hyporeflective state, which offered a clearer visualization of the transition between HFL and OPL. HFL accounted for an average 58% ± 3% of this thickness at a position 1 mm away from the foveal center in these eyes, with no significant difference between nasal and temporal HFL.

Manual segmentation of HFL along the entire extent of single-averaged B-scans obtained with Bioptigen in three subjects illustrated the difference between measurements of the entire thickness between the ELM and the OPL compared with the thickness of the ONL alone with the exclusion of HFL (Fig. 4). True ONL area represented 50%, 55%, and 49% of the area measured using the entire ELM to OPL space in subjects A, B, and C, respectively.

Two patients imaged with Bioptigen, each with macular pathology deforming the RPE and overlying retinal structures, were retrospectively identified. The first patient had a dome-like pigment epithelial detachment from central serous retinopathy (CSR). The second patient had age-related macular degeneration (AMD) with drusen causing an elevation of the RPE. Both of these pathologies had the effect of altering the angle at which HFL lies relative to the pupil. Variable reflectivity was evident in the anatomic space corresponding to HFL (Fig. 5), which appeared hyporeflective on the up-sloping aspect of the lesions closest to the foveal center while appearing hyperreflective on the down-sloping aspect of the pathology away from the foveal center. Two patients with macular pathology from CSR and AMD were also imaged dynamically using Cirrus. Significant changes in HFL intensity were observed at each SD-OCT beam entry position but were most striking at the extremes of the horizontal entry positions (Fig. 6).

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 (Fig. 7).

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 Figure 1 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 Figure 3, 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 Figures 5 and ​and66 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 Figure 6 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 Figure 4 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.