Revealing Henle’s Fiber Layer Using Spectral Domain
Optical Coherence Tomography
Brandon J. Lujan,1,2Austin Roorda,1Robert W. Knighton,3and Joseph Carroll4,5,6
PURPOSE. Spectral domain optical coherence tomography (SD-
OCT) uses infrared light to visualize the reflectivity of struc-
tures of differing optical properties within the retina. Despite
their presence on histologic studies, traditionally acquired SD-
OCT images are unable to delineate the axons of photorecep-
tor nuclei, Henle’s fiber layer (HFL). The authors present a new
method to reliably identify HFL by varying the entry position of
the SD-OCT beam through the pupil.
METHODS. Fifteen eyes from 11 subjects with normal vision
were prospectively imaged using 1 of 2 commercial SD-OCT
systems. For each eye, the entry position of the SD-OCT beam
through the pupil was varied horizontally and vertically. The
reflectivity of outer retinal layers was measured as a function of
beam position, and thicknesses were recorded.
RESULTS. The reflectivity of HFL was directionally dependent and
increased with eccentricity on the side of the fovea opposite the
entry position. When HFL was included in the measurement, the
thickness of the outer nuclear layer (ONL) of central horizontal
B-scans increased by an average of 52% in three subjects quanti-
fied. Four cases of pathology, in which alterations to the normal
macular geometry affected HFL intensity, were identified.
CONCLUSIONS. The authors demonstrated a novel method to
distinguish HFL from true ONL. An accurate measurement of
the ONL is critical to clinical studies measuring photoreceptor
layer thickness using any SD-OCT system. Recognition of the
optical properties of HFL can explain reflectivity changes im-
aged in this layer in association with macular pathology. (In-
vest Ophthalmol Vis Sci. 2011;52:1486–1492) DOI:10.1167/
pedicles and spherules that synapse in the retinal outer plexi-
enle’s fiber layer (HFL) contains bundles of unmyelinated
cone and rod photoreceptor axons terminating in the
form layer (OPL).1These fibers are intermingled with Mu ¨ller
cell processes and are obliquely oriented as a result of foveal
pit development where photoreceptors migrate inward and
ganglion cells migrate outward.2,3Like axons elsewhere in the
central nervous system, the axons of HFL contain microtubules
and are long, cylindrical structures.1–3Their average length is
558 ?m,4and the first synapses occur with dendrites of bipolar
and horizontal cells approximately 350 ?m from the foveal
center.4Given the large number of central foveal photorecep-
tor nuclei and this marked displacement, HFL constitutes a
significant fraction of the thickness of retinal layers within the
macula, as is evident histologically (Fig. 1). HFL is oriented
radially about the fovea and is indirectly visible ophthalmo-
scopically in patients with macular star formation in neuroreti-
nitis.5HFL also demonstrates the optical property of form
birefringence, a property that can be exploited to infer the
location of the foveal center as a direct consequence of its
consistent effects on polarized light.6
Optical coherence tomography (OCT) uses infrared light to
interferometrically derive optical reflectivity information vary-
ing by depth in living tissues.7The use of broadband light
sources, a spectrometer, and the application of signal process-
ing techniques has culminated in commercial spectral domain
(SD) OCT systems that are capable of imaging with a 5-?m axial
resolution in retinal tissue.8,9These improvements, along with
acquisition speeds fast enough to permit frame averaging with-
out 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.11However, 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).12–14Furthermore, 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.15–19Al-
though 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 norma-
tive 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.20In our study we describe a system-
atic method that can be applied to commercial SD-OCT sys-
tems to directly visualize and quantify HFL. Additionally, we
provide an optical explanation of this phenomenon and dem-
onstrate its clinical relevance.
From the1Department of Vision Science, School of Optometry,
University of California, Berkeley, California; the2West Coast Retina
Medical Group, San Francisco, California; the
Institute, University of Miami, Miller School of Medicine, Miami, Flor-
ida; and the Departments of4Ophthalmology,5Cell Biology, Neurobi-
ology, and Anatomy, and6Biophysics, Medical College of Wisconsin,
Presented in part at the annual meeting of the Association for
Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May
Supported by National Institutes of Health Grants K12 EY017269
(BJL), EY014375 (AR), P30EY001931 (JC) and R01EY017607 (JC); an
unrestricted departmental grant and a Career Development Award
from Research to Prevent Blindness (JC); and the Thomas M. Aaberg Sr.
Retina Research Fund (JC).
Submitted for publication May 25, 2010; revised September 2,
2010; accepted October 5, 2010.
Disclosure: B.J. Lujan, Carl Zeiss Meditec, Inc. (F); A. Roorda,
Carl Zeiss Meditec, Inc. (F); R.W. Knighton, Carl Zeiss Meditec, Inc.
(C); J. Carroll, None
Corresponding author: Brandon J. Lujan, Roorda Laboratory, 485
Minor Hall, Berkeley, CA 94720; email@example.com.
3Bascom Palmer Eye
Investigative Ophthalmology & Visual Science, March 2011, Vol. 52, No. 3
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
MATERIALS AND METHODS
All subjects provided informed consent to participate in this study.
This study was approved by Institutional Review Boards at the Univer-
sity of California at Berkeley and the Medical College of Wisconsin, and
the study protocol followed the tenets of the Declaration of Helsinki.
One or both eyes of each subject were dilated using 2.5% phenyl-
ephrine and 1% tropicamide. Two commercially available SD-OCT
systems were used to collect data for this study. Twelve eyes of eight
subjects and two eyes of two patients with macular disease were
imaged using Cirrus HD-OCT (Carl Zeiss Meditec, Inc., Dublin, CA) by
the same operator (BJL), and three eyes of three subjects were imaged
using a single Bioptigen SD-OCT (Bioptigen, Durham, NC) by a single
operator (JC). All scans for a given subject were performed during a
single session. The specific protocols for each device are described.
Additionally, a retrospective review of two clinical cases obtained
using Bioptigen SD-OCT was also performed.
Subjects ranged from 20 to 43 years of age and had refractive errors
ranging from a spherical equivalent of ?0.50 to ?4.50 diopters. SD-
OCT data were reviewed, and each included eye defined as normal was
determined to be free of macular pathology by a retinal specialist.
The Cirrus HD-OCT is an SD-OCT device that uses a 50-nm band-
width light source centered at 840 nm, has a 5-?m axial resolution, and
obtains 27,000 axially oriented scans (A-scans) per second. Cirrus data
are acquired over a 20° field (6 mm for an emmetropic eye) and has a
scan depth of 2 mm. Two standard Cirrus scan protocols were used to
acquire data—a 512 ? 128 cube and a frame-averaged cross-sectional
scan through the fovea. The 512 ? 128 cube consisted of 128 cross-
sectional images, or B-scans, that were each composed of 512 A-scans.
The frame-averaged cross-sectional scans were obtained using the
system’s commercial software (version 4.5), which averages 20 indi-
vidual B-scans each consisting of 1024 A-scans where the nominal
spacing between scans was set to zero. The rendered B-scans have an
aspect ratio of 2:1 (i.e., the scale is doubled in the axial dimension), but
this was corrected to 1:1 before data analysis described below.
Cirrus SD-OCT has a dedicated pupil camera that permits live
monitoring of the pupil position during scans and that indicates the
entry position of the SD-OCT beam. This was used in conjunction with
B-scans rendered in real time to obtain a beam entry position in which
both the horizontal and the vertical B-scans appeared “flat” (i.e., sym-
metric about the fovea.) Subsequently, the entrance pupil was dis-
placed at multiple intervals superiorly, inferiorly, temporally, and na-
sally. At each entrance pupil location, a horizontal and vertical frame
averaged cross-sectional scan was obtained. The absolute beam entry
position was documented using the pupil camera.
The Bioptigen SD-OCT used a 186.3-nm bandwidth centered at
878.4 nm, had a theoretical 1.4-?m axial resolution, and obtained
20,000 A-scans per second. Forty B-scans centered on the fovea, each
consisting of 1000 A-scans, were registered and averaged as previously
described.21A headrest was used to stabilize subjects, and deviation of
the entry position from where the B-scan appeared flat was measured
using markings on the stage. Additionally, video was recorded of live
vertical translation of the stage while vertically oriented B-scans were
Foveal fixation was confirmed by the midpoint position of the
foveal center in each analyzed B-scan. Scans that demonstrated low
signal strength or reduced image quality through frame averaging were
excluded. Volumetric data sets were evaluated immediately after ac-
quisition and were repeated if motion artifacts were present.
Cirrus B-scans were exported as bitmaps and analyzed using graphics
editing software (Photoshop CS4; Adobe Systems, Inc., San Jose, CA)
and ImageJ software (developed by Wayne Rasband, National Institutes
of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.
html). B-scan intensities were linearly normalized to assign the bright-
est pixel a value of white and the darkest pixel a value of black.
Measuring HFL Thickness
Horizontal frame-averaged B-scans acquired with Cirrus at the two
extreme horizontal entrance positions were used to measure the con-
tribution of different zones of reflectivity between the external limiting
membrane (ELM) and the inner plexiform layer (IPL) in five right eyes.
Specifically, the side of the B-scan that allowed full visualization of the
transition to the outer plexiform layer (OPL) was used for this analysis
where the inner segment/outer segment (IS/OS) was 1 mm from the
foveal center. The distance from the ELM to the posterior edge of the
OPL and the distance from the ELM to the edge of the observed
hyporeflective zone within this layer was measured along a line per-
pendicular to the IS/OS. The relative contribution of this hyporeflec-
tive zone corresponding to HFL was reported as a percentage of the
total distance between the ELM and the OPL.
Frame-averaged B-scans obtained with Bioptigen from each subject
were exported to Image J, where a manual tool was used to segment
the images collected at each of three pupil entry positions (nasal,
center, temporal). Segmented layer data were processed through cus-
tom computing software (MatLab; MathWorks, Natick, MA) to deter-
mine the thickness between the ELM and the outer edge of the OPL at
each eccentricity and between the ELM and the hyporeflective layer
corresponding to HFL.
Measuring Dependence of HFL Reflectance on
SD-OCT Scan Angle
Cirrus data were analyzed to assess the relative intensity of HFL as a
function of pupil entry positioned in six normal left eyes. The axial
ogy, courtesy of Roger C. Wagner, Pro-
fessor Emeritus of Biological Sciences,
University of Delaware, http://dspace.
dicated by the rectangle, showing the
and axons running in HFL. GCL, gan-
glion cell layer; PRs, photoreceptor IS
Mammalian foveal histol-
IOVS, March 2011, Vol. 52, No. 3
Revealing Henle’s Fiber Layer using SD-OCT1487
magnification of rendered Cirrus B-scans was corrected to display true
anatomic dimensions. The foveal center was defined as the point along
the photoreceptor IS/OS junction that was the greatest distance away
from the RPE along a line normal to the RPE. The angle between the
RPE and horizontal was measured 1 mm nasally and 1 mm temporally
to the foveal center on each B-scan along the horizontal meridian, with
a positive angle corresponding to a clockwise tilt. In each of these
B-scans, the average intensity and standard deviations of pixels con-
tained within a 500 ? 20-?m rectangle were measured. The rectangle
was oriented such that the long axis was parallel to the RPE. It was
centered horizontally 1 mm away from the fovea in both nasal and
temporal directions and was centered vertically within the inner as-
pect of the region between the ELM and the OPL corresponding to
HFL. Additionally, pixels contained within the same-sized rectangle
were obtained from the IPL anterior to the location of the first mea-
surement. The variance of IPL measurements with the pupil entry
position was found to be negligible; consequently, these served as a
normalization value against changes in incident intensity.
Movement of the SD-OCT beam entrance position horizontally
across the pupil was found to result in an apparent tilt of
horizontally acquired B-scans and resulted in a reflectivity
change corresponding anatomically to HFL. The alteration of
HFL reflectivity could be visualized on individual central hori-
zontal B-scans of the 512 ? 128 cube, but frame-averaged
B-scans augmented the effect through an improved signal-to-
noise ratio (Fig. 2). Changing the horizontal entry positions
while acquiring vertical B-scans did not result in any change of
reflectivity in the HFL, nor did it result in an apparent tilting of
the scan. Conversely, vertical translation of the beam entrance
position resulted in apparent tilting of vertically acquired B-
scans (Supplementary Movie S1, http://www.iovs.org/lookup/
suppl/doi:10.1167/iovs.10-5946/-/DCSupplemental) but not of
horizontally acquired B-scans (data not shown).
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 deter-
mining pupil entry position than relying on the pupil camera,
where image distortions increased the likelihood of registra-
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. Concur-
rently, 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 trans-
mission 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. Normal-
ization by the IPL resulted in a direct relationship between scan
angle and HFL intensity and demonstrated its directional reflec-
tance properties (Fig. 3). Reflectivity of the nasal macula in-
tally varying pupil entry position on
horizontal and vertical B-scans in a
right eye. (A) Central entry position
with flat B-scans and poor visualiza-
tion of HFL. Pupil entry position is
noted by central white x, not the
inferior corneal reflection. (B) Tem-
poral displacement of the entry
beam. Although the foveal anatomy
has not changed, there is an apparent
tilt to the horizontal B-scan because
of unequal optical path lengths (see
Fig. 6). A temporal entry position re-
sults in hyperreflectivity of HFL rela-
tive to the ONL nasal to the fovea
(black dots) and hyporeflectivity of
HFL temporal to the fovea (white
dots), where distinct demarcations
among the ONL, HFL, and IPL are
apparent. (C) Nasal displacement
of the entry beam showing the op-
The effects of horizon-
posite effects of B. (D–F) Vertical scans at each pupil entry position show only minimal HFL reflectivity changes and no tilt because vertically
the optical path lengths remain equal despite horizontal translation.
IPL reflectivity compared with the retinal angle measured 1 mm away
from the foveal center in six left eyes. Different symbols represent
individual subjects in nasal (black) and temporal (gray) macula.
Relative intensities of HFL reflectivity normalized by the
1488Lujan et al.
IOVS, March 2011, Vol. 52, No. 3
creased as scan angle decreased, which corresponded to tem-
poral 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 sub-
jects 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 reti-
nopathy (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 reflec-
tivity 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 appear-
ing hyperreflective on the down-sloping aspect of the pa-
thology away from the foveal center. Two patients with
macular pathology from CSR and AMD were also imaged
dynamically using Cirrus. Significant changes in HFL inten-
sity were observed at each SD-OCT beam entry position but
were most striking at the extremes of the horizontal entry
positions (Fig. 6).
In vivo cross-sectional retinal anatomy and pathology as visu-
alized 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
retinal thickness at each eccentricity along a single horizontal B-scan in
three subjects imaged on the Bioptigen SD-OCT. Dashed gray lines:
thickness measurements of the ONL using existing measurements of
Manual segmentations demonstrating the difference in
thickness between the ELM and the OPL. Solid black line: true mea-
surement of the ONL after removal of HFL.
IOVS, March 2011, Vol. 52, No. 3
Revealing Henle’s Fiber Layer using SD-OCT1489
of reflected light varies with pupil entry position, HFL exhibits
directional reflectance. We confirmed this finding was a con-
sistent effect using a variety of scan protocols, multiple sub-
jects, and two separate commercially available SD-OCT sys-
tems. We found this effect to be accentuated by, but not
dependent on, the use of frame averaging to reduce speckle
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 posi-
tion 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 sub-
jects 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 de-
gree 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 normal-
ized by the IPL in all subjects analyzed. The IPL was found to
be relatively invariant in reflectivity at each of the eccentrici-
ties 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 reti-
nal structures in which the optical principles behind their
occurrences are well understood. For example, in photorecep-
tor IS and OS, the optical Stiles-Crawford effect22exists be-
cause 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 experi-
ments.23,24The 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 dis-
cussed.25The mechanism of the directional reflectivity dis-
played by HFL was most consistent with that of the RNFL
the reflectivity visible in HFL. (A) HFL temporal to drusen appears
hyperreflective when acquired with a nasal beam entry. (B) Temporal
beam entry alters this intensity significantly. (C, D) Variation in HFL
intensity is seen to vary with beam position in this patient with
subretinal fluid from central serous retinopathy.
Clinical examples of the effect of beam entry position on
reflectivity changes in HFL. (A) Cen-
tral serous chorioretinopathy. Imme-
diately above the serous pigment ep-
ithelial detachment, HFL reflectivity
varies from hyporeflective (white
dots) to hyperreflective (black dots)
based on the RPE geometry altering
the orientation of HFL. Note the
changes in the IS/OS junction reflec-
tivity and the homogeneous reflectiv-
ity of the IPL. (B) Nonexudative (dry)
AMD. Because of drusen there is RPE
elevation that alters the orientation of
HFL and produces similar hyporeflec-
tive and hyperreflective segments.
Clinical visualization of
1490 Lujan et al.
IOVS, March 2011, Vol. 52, No. 3
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 im-
aged 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 horizon-
tally 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 shal-
lower angles of incidence, light scattered further away from
the exit pupil, creating a hyporeflective HFL on SD-OCT
Quantitative estimates suggest results that are consistent
with our observations. Using the Bennett-Rabbetts’ model
eye,26the approximate angle of incidence of light rays ema-
nating 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 reflectiv-
ity 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 reflec-
tivity provides an explanation for the “unexpected” reflectivity
in this layer because of pathology that affects the retinal geom-
etry. The images in Figures 5 and 6 demonstrate reflectivity
changes caused by alterations in the normal geometry of the
retina, where pigment epithelial detachment, drusen, or sub-
retinal fluid alters the angle of the cone of light reflecting from
HFL. Optical changes in HFL are introduced by these protuber-
ances 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 the-
orized to represent a “degenerative cellular process.”19Al-
though 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
Identification of the true dimensions of the ONL is critical to
clinical studies that aim to accurately measure macular photo-
receptor 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.19might 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 density27and foveal pit
This study did not seek to convey average or normal values
of the HFL and ONL thicknesses. Accurate reporting of norma-
tive 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. Further-
more, it is unclear whether HFL intensity and thickness mea-
surements 1 mm away from the foveal center are more impor-
tant 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 inten-
sity 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
tric 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 retina) and
scatter at an angle equal to its angle of incidence. A light ray along path
A (gray line) scatters back from HFL close to the normal and back
through the pupil to produce a hyperreflective HFL. However, a light
ray along path B (black) scatters toward the opposite side of the eye
without exiting the pupil and consequently appears hyporeflective on
a corresponding B-scan. Note path A is longer than path B, accounting
for the tilt of the B-scans in Figure 2.
Unscaled schematic of HFL reflectivity through an eccen-
IOVS, March 2011, Vol. 52, No. 3
Revealing Henle’s Fiber Layer using SD-OCT1491
retinal tissue. Importantly, an SD-OCT image depends not only Download full-text
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.
The authors thank Jacque L. Duncan and Adam M. Dubis for technical
1. Hogan MJ, Alvarado J, Weddell J. Histology of the Human Eye.
Philadelphia: WB Saunders Company; 1971.
2. Hendrickson AE, Yuodelis C. The morphological development of
the human fovea. Ophthalmology. 1984;91:603–612.
3. Yuodelis C, Hendrickson A. A qualitative and quantitative analysis
of the human fovea during development. Vision Res. 1986;26:847–
4. Drasdo N, Millican CL, Katholi CR, Curcio CA. The length of Henle
fibers in the human retina and a model of ganglion receptive field
density in the visual field. Vision Res. 2007;47:2901–2911.
5. Gass JDM. Stereoscopic Atlas of Macular Diseases. 3rd ed. St.
Louis, MO: CV Mosby Company; 1987:746–751.
6. Hunter DG, Patel SN, Guyton DL. Automated detection of foveal
fixation by use of retinal birefringence scanning. Appl Opt. 1999;
7. Puliafito CA, Hee MR, Lin CP, et al. Imaging of macular diseases
with optical coherence tomography. Ophthalmology. 1995;102:
8. Sull AC, Vuong LN, Price LL, et al. Comparison of spectral/Fourier
domain optical coherence tomography instruments for assessment
of normal macular thickness. Retina. 2010;30:235–245.
9. Wojtkowski M, Leitgeb R, Kowalczyk A, Bajraszewski T, Fercher
AF. In vivo human retinal imaging by Fourier domain optical
coherence tomography. J Biomed Opt. 2002;7:457–463.
10. Jorgensen TM, Thomadsen J, Christensen U, Soliman W, Sander B.
Enhancing the signal-to-noise ratio in ophthalmic optical coher-
ence tomography by image registration–method and clinical ex-
amples. J Biomed Opt. 2007;12:041208.
11. Anger EM, Unterhuber A, Hermann B, et al. Ultrahigh resolution
optical coherence tomography of the monkey fovea: identification
of retinal sublayers by correlation with semithin histology sec-
tions. Exp Eye Res. 2004;78:1117–1125.
12. Ko TH, Fujimoto JG, Schuman JS, et al. Comparison of ultrahigh-
and standard-resolution optical coherence tomography for imag-
ing macular pathology. Ophthalmology. 2005;112:23.
13. Ergun E, Hermann B, Wirtitsch M, et al. Assessment of central
visual function in Stargardt’s disease/fundus flavimaculatus with
ultrahigh-resolution optical coherence tomography. Invest Oph-
thalmol Vis Sci. 2005;46:310–316.
14. Matsumoto H, Sato T, Kishi S. Outer nuclear layer thickness at the
fovea determines visual outcomes in resolved central serous cho-
rioretinopathy. Am J Ophthalmol. 2009;148:105–110.
15. Christensen UC, Kroyer K, Thomadsen J, Jorgensen TM, la Cour M,
Sander B. Normative data of outer photoreceptor layer thickness
obtained by software image enhancing based on Stratus optical
coherence tomography images. Br J Ophthalmol. 2008;92:800–
16. Bagci AM, Shahidi M, Ansari R, Blair M, Blair NP, Zelkha R. Thick-
ness profiles of retinal layers by optical coherence tomography
image segmentation. Am J Ophthalmol. 2008;146:679–687.
17. DeBuc DC, Somfai GM, Ranganathan S, Tatrai E, Ferencz M, Pu-
liafito CA. Reliability and reproducibility of macular segmentation
using a custom-built optical coherence tomography retinal image
analysis software. J Biomed Opt. 2009;14:064023.
18. Rha J, Dubis AM, Wagner-Schuman M, et al. Spectral domain
optical coherence tomography and adaptive optics: imaging pho-
toreceptor layer morphology to interpret preclinical phenotypes.
Adv Exp Med Biol. 2010;664:309–316.
19. Schuman SG, Koreishi AF, Farsiu S, Jung SH, Izatt JA, Toth CA.
Photoreceptor layer thinning over drusen in eyes with age-related
macular degeneration imaged in vivo with spectral-domain optical
coherence tomography. Ophthalmology. 2009;116:488–496.
20. Byeon SH, Chu YK. Interpretation of fovea center morphologic
features in optical coherence tomography. Am J Ophthalmol.
21. Tanna H, Dubis AM, Ayub N, et al. Retinal imaging using commer-
cial broadband optical coherence tomography. Br J Ophthalmol.
22. Gao W, Cense B, Zhang Y, Jonnal RS, Miller DT. Measuring retinal
contributions to the optical Stiles-Crawford effect with optical
coherence tomography. Opt Express. 2008;16:6486–6501.
23. Knighton RW, Baverez C, Bhattacharya A. The directional reflec-
tance of the retinal nerve fiber layer of the toad. Invest Ophthal-
mol Vis Sci. 1992;33:2603–2611.
24. Knighton RW, Huang XR. Directional and spectral reflectance of
the rat retinal nerve fiber layer. Invest Ophthalmol Vis Sci. 1999;
25. Knighton RW, Qian C. An optical model of the human retinal
nerve fiber layer: implications of directional reflectance for vari-
ability of clinical measurements. J Glaucoma. 2000;9:56–62.
26. Bennet AG, Rabbetts RB. Bennett and Rabbett’s Clinical Visual
Optics. 3rd ed. Edinburgh, UK: Butterworth-Heinemann; 1998:
27. Curcio CA, Sloan KR, Kalina RE, Hendrickson AE. Human photo-
receptor topography. J Comp Neurol. 1990;292:497–523.
28. Dubis AM, McAllister JT, Carroll J. Reconstructing foveal pit mor-
phology from optical coherence tomography imaging. Br J Oph-
1492 Lujan et al.
IOVS, March 2011, Vol. 52, No. 3