Reproducibility of Spectral-Domain Optical Coherence
Tomography Total Retinal Thickness Measurements
Michelle L. Gabriele,1,2,3Hiroshi Ishikawa,1,2Joel S. Schuman,1,2,3Richard A. Bilonick,1
Jongsick Kim,1,2Larry Kagemann,1,2and Gadi Wollstein1
PURPOSE. To test the reproducibility of spectral-domain optical
coherence tomography (SD-OCT) total retinal thickness (TRT)
measurements in mice.
METHODS. C57Bl/6 mice were anesthetized, and three repeated
volumetric images were acquired in both eyes with SD-OCT
(250 A-scans ? 250 frames ? 1024 samplings), centered on the
optic nerve head (ONH). The mice were repositioned between
scans. TRT was automatically measured within a sampling band
of retinal thickness with radii of 55 to 70 pixels, centered on
the ONH by using custom segmentation software. The first
volumetric image acquired in a given eye was used to register
the remaining two SD-OCT images by manually aligning the en
face images with respect to rotation and linear translation.
Linear mixed-effects models were fitted to global and quadrant
thicknesses, taking into account the clustering between eyes,
to assess imprecision (measurement reproducibility).
RESULTS. Twenty-six eyes of 13 adult mice (age 13 weeks)
were imaged. The mean global TRT across all eyes was
298.21 ?m, with a mouse heterogeneity standard deviation
(SD) of 4.88 ?m (coefficient of variation [CV] ? 0.016), an
eye SD of 3.32 ?m (CV ? 0.011), and a device-related impre-
cision SD of 2.33 ?m (CV ? 0.008). The superior quadrant had
the thickest mean TRT measurement (310.38 ?m) and the
highest (worst) imprecision SD (3.13 ?m; CV ? 0.010), and
the inferior quadrant had the thinnest mean TRT (291.55 ?m).
The quadrant with the lowest (best) imprecision SD was in the
nasal one (2.06 ?m; CV ? 0.007).
CONCLUSIONS. Good reproducibility was observed for SD-OCT
retinal thickness measurements in mice. SD-OCT may be useful
for in vivo longitudinal studies in mice. (Invest Ophthalmol Vis
Sci. 2010;51:6519–6523) DOI:10.1167/iovs.10-5662
humans, retinal thickness and retinal nerve fiber layer thick-
ness measurements obtained with OCT have been shown to be
reproducible,1–10and this has made OCT valuable for cross-
sectional and longitudinal studies.11–13
In mice, time-domain OCT has been used to obtain cross-
sectional images of the retina in vivo.14–16Spectral-domain
(SD)-OCT, which has faster scanning rates and higher axial
resolution, allows the acquisition of detailed three-dimensional
(3-D) volumetric scans in mice.17–25Although SD-OCT acquires
volumetric information in mice, thickness measurements ob-
tained post hoc were often based on single cross sections.19,23
In considering the use of SD-OCT for the longitudinal fol-
low-up of mouse models of retinal diseases, however, a con-
sistent method for acquiring 3-D data must be developed. The
goal of this study was to evaluate the reproducibility of a
SD-OCT method of measuring total retinal thickness (TRT) in
ptical coherence tomography (OCT) permits rapid, non-
invasive, in vivo quantification of retinal structures. In
This experiment was approved by the University of Pittsburgh’s Insti-
tutional Animal Care and Use Committee and adhered to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Healthy adult male C57Bl/6 mice (Jackson Laboratory, Bar Harbor, ME)
were used in the study. The mice were maintained in the University of
Pittsburgh Animal Facility in a 12-hour light/dark cycle and had free
access to water and standard laboratory feed.
The mice were anesthetized with an intraperitoneal injection of keta-
mine (80 mg/kg; Ketaject; Phoenix Pharmaceuticals, St. Joseph, MO)
and xylazine (5 mg/kg; Xyla-ject; Phoenix Pharmaceuticals), to prevent
large movements during SD-OCT image acquisition. Both pupils were
dilated with a topically applied drop of tropicamide (1%; Falcon Phar-
maceuticals, Fort Worth, TX). To neutralize corneal optical power and
focus the SD-OCT beam onto the retina, we applied a thin glass
coverslip to the cornea and used hydroxymethylcellulose ophthalmic
demulcent solution (Goniosol 2.5%; Akorn, Buffalo Grove, IL) to pre-
serve corneal hydration and couple the coverslip to the cornea. The
mice were secured on a custom stage (Fig. 1), which allowed free
rotation, to align the eye for imaging of the optic nerve head (ONH).
Three repeated volumetric images, centered on the ONH, were
acquired in both eyes with SD-OCT (Bioptigen, Inc., Durham, NC). All
SD-OCT images consisted of 250 averaged A-scans (each A-scan was an
From the1UPMC Eye Center, Eye and Ear Institute, Ophthalmol-
ogy and Visual Science Research Center, Department of Ophthalmol-
ogy, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylva-
nia; the2Department of Bioengineering, Swanson School of Engineering,
University of Pittsburgh, Pittsburgh, Pennsylvania; and the3Center for the
Neural Basis of Cognition, Carnegie Mellon University and University of
Pittsburgh, Pittsburgh, Pennsylvania.
Presented in part at the 2010 annual meeting of the International
Society for Imaging in the Eye, Fort Lauderdale, Florida, May 2010.
Supported in part by National Institutes of Health Grants T32-
EY017271, R21-EY019092, R01-EY013178, and P30-EY008098 (Be-
thesda, MD); the Eye and Ear Foundation (Pittsburgh, PA); and Re-
search to Prevent Blindness (New York, NY).
Submitted for publication April 7, 2010; revised June 4, 2010;
accepted June 11, 2010.
Disclosure: M.L. Gabriele, None; H. Ishikawa, P; J.S. Schuman,
P; R.A. Bilonick, None; J. Kim, None; L. Kagemann, None; G.
Wollstein, Carl Zeiss Meditec, Optovue (F), P.
Corresponding author: Joel S. Schuman, UPMC Eye Center, 203
Lothrop Street, Eye and Ear Institute, Room 816, Pittsburgh, PA 15213;
Investigative Ophthalmology & Visual Science, December 2010, Vol. 51, No. 12
Copyright © Association for Research in Vision and Ophthalmology
average of four A-scans), 250 frames, and 1024 samplings in depth.
These parameters correspond to a volume of approximately 1.5 ?
1.5 ? 2.0 mm at the surface of the coverslip. The mice were reposi-
tioned between scans by rotating the stage (when switching between
left and right eyes) or removing the mouse from the stage for complete
repositioning (when returning to same eye). Care was taken to ensure
that all three scans were acquired before the mice developed revers-
ible cataract. Images were acquired within 20 minutes after anesthesia
SD-OCT Image Analysis: Manual Alignment
Repositioning the mouse between scans resulted in variability in the
location and orientation of the ONH. We manually aligned the SD-OCT
en face images after acquisition (Fig. 2) to compensate for the variabil-
ity in orientation. The first SD-OCT en face image acquired in a given
eye was used as an alignment reference for subsequent images, and the
coordinates of rotation and translation for each scan relative to the
reference were used to align the thickness measurements.
SD-OCT Image Analysis: Automated Segmentation
The ONH margin for each eye was manually located by one experi-
enced operator (MG) on each OCT en face image. The geometric
center of the ONH margin was then used as a center point for subse-
quent analysis. TRT was automatically measured within a sampling
band of retinal thickness with an inner radius of 55 pixels and an outer
radius of 70 pixels, centered on the ONH, by using custom segmenta-
tion software to detect the internal limiting membrane and retinal
pigment epithelium (Fig. 3).26This sampling band was chosen to avoid
regions within the optic nerve and areas of vignetting, as automated
segmentation tends to be less reliable in those regions. After the
sampling band thickness information was obtained, the scans were
rotated and translated on the basis of the coordinates obtained from
manual image alignment. These aligned thickness measurements were
used to assess reproducibility. It should be noted that alignment was
completely independent of thickness measurements.
SD-OCT Image Analysis: Quality Criteria
SD-OCT en face images were checked to ensure that there was con-
sistent image quality across the scan and that there were no areas of
shadowing from media opacities within the TRT sampling band, as it
could affect thickness measurements. The eyes were excluded if the
TRT segmentation algorithm failed at any location inside the sampling
band. Algorithm failure was defined as a clear disruption of the border,
either the inner limiting membrane (ILM) or RPE, for more than five
consecutive A-scans within the sampling band. If any one of the three
scans per eye had to be excluded, both eyes of the mouse were
excluded from the analysis.
Linear mixed-effects models were fitted to global and quadrant TRTs,
taking into account the clustering between eyes, to assess measure-
ment reproducibility. Imprecision (as measured by the residual stan-
dard deviation [SD]), and corresponding 95% confidence intervals
were computed. All analysis was conducted with R Language and
Environment for Statistical Computing program (http://www.R-project.
org).27An ? level of 0.05 was used as the cutoff for statistical
views of the stage used for alignment
of the mouse for SD-OCT imaging of
the ONH. A coverslip was used to
account for the steep curvature of
the mouse cornea.
Side (left) and top (right)
Reference SD-OCT en face image and (B) subsequent SD-OCT en face
image. (C) Scan registered to the reference.
Manual alignment of two SD-OCT en face images. (A)
the location of the OCT B-scan (right). The B-scan demonstrates auto-
mated segmentation of the ILM (white line) and RPE (gray line) to
obtain measurements. Vertical lines: disc margin.
SD-OCT en face image (left) with the black line indicating
6520 Gabriele et al.
IOVS, December 2010, Vol. 51, No. 12
The mixed-effects model for TRT (yijk) for mouse i, eye k nested
within mouse i, and replicate k is:
yijk? ? ? ai? bij? ?ijk
where ? represents the cohort mean TRT, aiis the random intercept
for mouse i, assumed to be normally distributed with a mean of 0 and
SD ?m[ai? N(0,?m
mouse i [bij? N(0,?e
[?ijk? N(0,?2)]. The standard deviations ?mand ?erepresent the
variability of the thickness measurements coming from the cohort of
mice (heterogeneity across mice and eye, respectively), whereas the
error SD represents the imprecision coming from the device. This
device-related imprecision includes errors coming from segmentation
2)]; bijis a random intercept for eye j nested within
2)]; and ?ijkis a normally distributed random error
algorithm performance as well as manual alignment of SD-OCT en face
Thirty eyes of 15 healthy adult male mice, age 13 weeks, were
imaged. Two eyes were excluded due to segmentation algo-
rithm failure: one at the level of the RPE and one at the ILM.
The contralateral eyes of those two mice were also excluded.
In total, 26 eyes of 13 healthy adult male mice were included.
Figure 4 shows an example of the alignment of TRT maps
relative to reference.
Mean global TRT across all eyes was 298.21 ?m (Table 1),
with a mouse heterogeneity SD of 4.88 ?m (CV ? 0.016) and
reference scan. (A, C, F) OCT en face
images; (B, D, G) TRT thickness map
with sampling region indicated as
the region between red concentric
circles. (E, H) TRT maps registered
to the reference.
maps aligned to a single
TABLE 1. Estimates of Mixed-Effect Model Parameters and CV for Global and Sectoral TRT
SectorEstimated Mean (?)
All estimates are in micrometers except for CV, which is unitless.
* Device reproducibility measurement.
IOVS, December 2010, Vol. 51, No. 12
Mouse OCT Reproducibility6521
an eye SD of 3.32 ?m (CV ? 0.011). The device-related global
imprecision SD was 2.33 ?m (CV ? 0.008). The superior
quadrant showed the thickest TRT measurements (310.38 ?m,
mouse heterogeneity SD, 6.09 ?m [CV ? 0.020], eye SD 6.98
?m [CV ? 0.022]), whereas the inferior quadrant was the
thinnest (291.55 ?m, mouse heterogeneity SD, 6.02 ?m [CV ?
0.021], eye heterogeneity SD, 8.13 ?m [CV ? 0.028]). The
highest (worst) quadrant imprecision SD was in the superior
quadrant (3.13 ?m; CV ? 0.010) and the lowest (best) was in
the nasal quadrant (2.06 ?m; CV ? 0.007).
In this study, we were able to demonstrate good intrasession
reproducibility of SD-OCT TRT measurements in mice that
were obtained with automated retinal segmentation software.
This study lays the foundation for future work monitoring
retinal changes in mice over time. The global imprecision
estimates were 2.33 ?m. Hence, a TRT difference between two
observations from the same eye of the same mouse of at least
6.46 ?m (2.33 ?m ? 1.96 ? ?2; 2.17% of the mean TRT; 3.31
pixels) would be necessary to conclude, with 95% confidence,
that the difference was due to an actual structural change as
opposed to variability inherent in the device.
Although the superior quadrant showed the worst quadrant
imprecision SD (3.13 ?m), the superior CV (0.010) was actu-
ally slightly better than that of the inferior quadrant (CV ?
0.011). This discrepancy is attributable to the differences in
thickness within those quadrants: the superior quadrant had
the thickest mean measurements, whereas the mean inferior
quadrant measurements were the thinnest (310.38 ?m vs.
291.55 ?m respectively). Overall, the CV was similar for the
superior, inferior, and temporal quadrants, but was lower in
the nasal quadrant (CV ? 0.007).
Our study was designed to evaluate intrasession reproduc-
ibility, as the mice were only anesthetized once and repeatedly
scanned in one session. In multiple studies of OCT imaging,
this source of variability was observed to be the main one.28,29
Intersession variability has been shown to add minimal vari-
ability and was therefore not the focus of this study.28
Our TRT measurements are thicker than those reported by
Horio et al.14They reported only temporal retinal thickness
from a single A-scan at a distance of 1 to 2 disc diameters from
the temporal margin of the optic disc. This distance was farther
from the disc center than we measured and therefore can be
expected to show a lesser retinal thickness. In addition, since
their measurement was based on a single A-scan, there may
have been wide variability in their measurements. They also
used a lower resolution OCT system as well as a different strain
(BALB/c) of older (16 weeks) mice, all of which may explain
the observed deviation. Li et al.15showed retinal thickness
measurements of 200 to 250 ?m in C57Bl/6 mice. Their mea-
surements were based on single cross sections, and they used
a lower resolution OCT system. Ruggeri et al.18measured
retinal thickness with SD-OCT and reported an average thick-
ness of 202 ?m. This average was based on all points outside of
a 0.5-mm-diameter region covering the ONH, whereas our
sampling location was within a band with a diameter of 55 to
70 pixels. We chose this sampling region to ensure that we
were sampling outside the ONH region and not extending into
a region where there could be vignetting of the image from the
pupil. Compared to our sampling region, their average in-
cluded thinner regions of the retina, which may explain the
difference in global measurements. In addition, differences in
measurements due to different devices with different segmen-
tation algorithms may partially explain this discrepancy. These
dissimilarities may also explain the slight differences in our
measurements compared with those reported by Kim et al.,19
Huber et al.,23Fischer et al.,24and Cebulla et al.25
Our observation of slightly thinner TRT measurements in-
feriorly was unexpected. One reason for this finding may be
that the orientation of the eye during scanning is not the true
orientation of the mouse, since the mouse was rotated on a
stage during acquisition, to allow for focusing on the ONH. The
rotation may have led to variability of the exact quadrant
boundary locations across eyes. This limitation is inherent in
our method of obtaining images centered on the ONH by
rotating the mouse on a stage.
Another limitation is that manual labeling of the optic disc
margin is necessary to obtain the geometric center of the disc.
It is possible, however, that automated detection of the ONH
(and ONH center) may be available in the future, similar that
currently used for detection in human eyes.30In addition, we
used manual registration of en face images to account for
rotational and translational eye position differences from scan
to scan. This process accounts for shifts that occurred between
scans, because our stage allowed for rotation and translation of
the eye to center the image on the ONH, and accounts for x-
and y-scan location differences. A stage that would allow for
more consistent alignment of mice between scans may elimi-
nate this step. It may also be possible to use automated regis-
tration based on blood vessel segmentation in en face images to
replace the manual component of the analysis. Future studies
investigating this possibility are necessary.
We have shown that SD-OCT allows reproducible 3D scan-
ning of tissue structure in vivo that is minimally invasive. As a
result, SD-OCT has the potential to reduce the number of mice
needed in studies of diseases that affect retinal structure by
allowing the same mice to be consistently observed from
session to session. Therefore, longitudinal studies monitoring
one population of mice are possible, as opposed to the current
approach of cross-sectional analysis with a subset of mice at
multiple time points. This method should allow researchers to
better observe the structural manifestations of disease over
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