Posterior scleral birefringence measured by triple-input polarization-sensitive imaging as a biomarker of myopia progression

Abstract

In myopic eyes, pathological remodelling of collagen in the posterior sclera has mostly been observed ex vivo. Here we report the development of triple-input polarization-sensitive optical coherence tomography (OCT) for measuring posterior scleral birefringence. In guinea pigs and humans, the technique offers superior imaging sensitivities and accuracies than dual-input polarization-sensitive OCT. In 8-week-long studies with young guinea pigs, scleral birefringence was positively correlated with spherical equivalent refractive errors and predicted the onset of myopia. In a cross-sectional study involving adult individuals, scleral birefringence was associated with myopia status and negatively correlated with refractive errors. Triple-input polarization-sensitive OCT may help establish posterior scleral birefringence as a non-invasive biomarker for assessing the progression of myopia.

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Nature Biomedical Engineering | Volume 7 | August 2023 | 986–1000 986
nature biomedical engineering
Article
https://doi.org/10.1038/s41551-023-01062-w
Posterior scleral birefringence measured by
triple-input polarization-sensitive imaging
as a biomarker of myopia progression
Xinyu Liu 1,2,3, Liqin Jiang1,2, Mengyuan Ke 1,4, Ian A. Sigal5,6,
Jacqueline Chua1,2,3, Quan V. Hoang1,2,7,8, Audrey WI. Chia1,2,
Raymond P. Najjar 1,2,7, Bingyao Tan1,3,9, Jocelyn Cheong 1,2,
Valentina Bellemo3,10, Rachel S. Chong1,2, Michaël J. A. Girard1,2,11, Marcus Ang1,2,
Mengyang Liu1,4, Gerhard Garhöfer12, Veluchamy A. Barathi2,7,1 3,
Seang-Mei Saw1,2,14, Martin Villiger 15 & Leopold Schmetterer 1,2, 3,4 ,9,1 0,11 ,12
In myopic eyes, pathological remodelling of collagen in the posterior
sclera has mostly been observed ex vivo. Here we report the development
of triple-input polarization-sensitive optical coherence tomography
(OCT) for measuring posterior scleral birefringence. In guinea pigs and
humans, the technique oers superior imaging sensitivities and accuracies
than dual-input polarization-sensitive OCT. In 8-week-long studies with
young guinea pigs, scleral birefringence was positively correlated with
spherical equivalent refractive errors and predicted the onset of myopia.
In a cross-sectional study involving adult individuals, scleral birefringence
was associated with myopia status and negatively correlated with refractive
errors. Triple-input polarization-sensitive OCT may help establish posterior
scleral birefringence as a non-invasive biomarker for assessing the
progression of myopia.
Myopia (near-sightedness) is a prevalent vision disorder that can be
corrected by eyeglasses, contact lenses or refractive surgery. How-
ever, unmitigated progression to high myopia exposes patients to an
increased risk of developing vision-threatening complications
1,2
. Recent
studies have reported that 10–30% of patients with high myopia develop
associated pathological complications later in life
3,4
, including myopic
maculopathy and optic neuropathy, which lead to irreversible visual
impairment5,6. Clinical interventions for retarding the progression
of early-stage myopia and rescuing eyes with pathological complica-
tions are available7,8. However, reliable biomarkers guiding the timing
of treatment are lacking. Specifically, for early-stage myopia, topical
atropine has been proved effective in controlling myopia progression
9
,
yet its adverse effects preclude universal application to all patients10,11.
Currently, treatment decisions are based on documented myopia
Received: 22 March 2022
Accepted: 30 May 2023
Published online: 26 June 2023
Check for updates
1Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Singapore. 2Academic Clinical Program, Duke-NUS Medical School,
Singapore, Singapore. 3SERI-NTU Advanced Ocular Engineering (STANCE) programme, Singapore, Singapore. 4Center for Medical Physics and
Biomedical Engineering, Medical University of Vienna, Vienna, Austria. 5Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA.
6Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA. 7Department of Ophthalmology, Yong Loo Lin School of Medicine National
University of Singapore, Singapore, Singapore. 8Department of Ophthalmology, Columbia University, New York, NY, USA. 9School of Chemistry, Chemical
Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore. 10Lee Kong Chian School of Medicine, Nanyang Technological
University, Singapore, Singapore. 11Institute of Molecular and Clinical Ophthalmology, Basel, Switzerland. 12Department of Clinical Pharmacology,
Medical University of Vienna, Vienna, Austria. 13Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, Singapore, Singapore.
14Saw Swee Hock School of Public Health, ,National University of Singapore, National University Health System, Singapore, Singapore. 15Wellman Center
for Photomedicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA. e-mail: leopold.schmetterer@ntu.edu.sg
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Article https://doi.org/10.1038/s41551-023-01062-w
PS-OCT instruments using sequential dual-input polarization states
41,42
assume that the measurements contain only pure retardance and that
the impact of sample diattenuation is negligible
43
. Depth-encoding
PS-OCT
44–46
can measure diattenuation but reduces the imaging range
due to multiplexing of the images of two polarization input states
along depth. TRIPS-OCT measures diattenuation and corrects for
depolarization while maintaining the simplicity of dual-input systems.
We demonstrated that TRIPS-OCT improved birefringence sensitivity
and accuracy of optic axis measurement compared with dual-input
PS-OCT. Moreover, using histological sections of the posterior sclera of
pig and guinea pig eyes, we validated in vivo TRIPS-OCT birefringence
imaging with PLM and TEM.
To examine PSB as a biomarker for myopia, first, we used a guinea
pig myopia model47 (42 eyes) to longitudinally evaluate the correlation
between PSB and development of refractive errors in animals of 2–8
weeks age. PSB measured at 2 weeks of age was an effective predictive
biomarker for the onset of myopia at the ages of 4 and 8 weeks, better
than baseline SE, which has been reported as the best single predictor
for myopia onset
48,49
. Next, in our human cross-sectional study, we
found a strong, albeit negative, correlation between PSB and myopia
status within eyes with emmetropia (normal vision) and low myopia
(69 eyes, −6D < SE ≤ 3D). To the best of our knowledge, there have
been no previous clinical studies focusing on PSB in patients with
myopia. Moreover, in patients with pathologic myopia, we observed
a spatial association of PSB with staphyloma. We next determined that
PSB was a better classifier than axial length to differentiate eyes with
pathologic myopia (15 eyes) from those with high myopia (16 eyes). In
eyes with high myopia, we found increased PSB to be associated with
the presence of peripapillary atrophy (PPA)
50,51
and to possibly indicate
an increased risk of progression to a more advanced stage of myopia.
Overall, in this study, we demonstrated the potential of PSB, measured
with TRIPS-OCT, as a predictive biomarker for myopia, from childhood
myopia development to late-life complications.
Results
Sensitivity and accuracy of TRIPS-OCT
We developed TRIPS-OCT (Extended Data Fig. 1) to address the chal-
lenge of performing reliable birefringence measurements in the clini-
cal setting. To demonstrate the improved birefringence sensitivity of
TRIPS-OCT compared with the dual-input reconstruction method, we
imaged a guinea pig retina in vivo (Fig. 1a) and reconstructed the local
birefringence images using the dual-input method and the proposed
method. In this comparison, we ensured that the sampling time of the
signals used by the two methods were identical (Extended Data Fig. 2a).
As the inner retina of the guinea pig exhibits low birefringence, distri-
butions of its birefringence (Fig. 1b) can approximate the characteris-
tics of the birefringence background noise. The standard deviation of
the noise, or noise floor, of TRIPS-OCT was 48% lower than that of the
conventional dual-input method (Fig. 1b and Extended Data Fig. 2b,c).
We observed that the dual-input reconstruction method suffers
from edge artefacts that are associated with variations in the sample
scattering profile (Extended Data Fig. 3a,b). The edge artefact (Sup-
plementary Discussion 1) is a dominant source of birefringence noise
that is induced by a shift in the point-spread-functions (PSF) due to
the polarization mode dispersion of preceding tissue layers, includ-
ing the cornea. Because this shift leads to apparent diattenuation, the
resulting artefacts are markedly suppressed by correctly accounting
for diattenuation in the Mueller matrices of the sample. TRIPS-OCT
isolates the sample retardance from the Mueller matrices, properly
separating the effects of polarization-dependent scattering, sample
diattenuation and apparent diattenuation, and thus is almost free from
edge artefacts (Extended Data Fig. 3c).
To test whether compensation for corneal retardance and dia-
ttenuation improves the accuracy of optic axis measurements, we
scanned the Henle’s fibre layer (HFL) in the retina of a healthy volunteer
progression
12
, that is, the baseline and deterioration of the spherical
equivalent refractive error (SE) in the past year. However, large fluctua-
tions in SE during myopia development in childhood and the scarcity
of documented records pose practical issues in decision making13. For
end-stage myopia, posterior scleral reinforcement (PSR) surgery, includ-
ing macular buckling, is a clinically available therapy to strengthen the
posterior sclera and arrest the continued elongation of the eye14,15. But
standards for whether and when to perform PSR surgeries are controver-
sial and inconclusive
15,16
. To guide treatment decision-making, there is a
compelling need for biomarkers that reliably predict myopia progres-
sion and indicate early pathological changes in myopic eyes.
Owing to its pivotal role in defining eye shape, the sclera has been
extensively studied in animal models and humans with myopia or path-
ologic myopia17–20. The sclera is a dense, collagen-rich and mechanically
strong tissue that coats the eye and protects its internal structures21.
During the development and progression of myopia, the posterior
segment of the sclera undergoes a remodelling process that includes
thinning22, weakening23 and enlargement in surface area24, resulting in
an excessive axial elongation of the eye that impairs its optical function.
Furthermore, extensive scleral remodelling may predispose patients to
staphyloma, an irregular outpouching of the posterior eyewall, which
is a defining characteristic of pathologic myopia. Staphyloma may
create shear forces across the retina and is one of the main pathophysi-
ological factors of myopia-associated vision-threatening complica-
tions25. At present, staphyloma is diagnosed through the observation
of an irregular eye shape using ultrasonography or wide-field optical
coherence tomography (OCT)6. However, eye-shape deformation may
be a secondary result of extensive scleral remodelling, which, by this
point, may already have caused irreversible retinal damage
26
. From the
early stages to the late stages of myopia, scleral collagen is constantly
remodelling at the microscopic level; these changes include a decrease
in collagen fibre diameter27,28, a shift towards disordered architecture
with a reduction in the number of interwoven fibres
22,29
and alterations
in fibre direction
30
. Imaging techniques including polarization light
microscopy (PLM)31,32 and transmission electron microscopy (TEM)
have been essential in identifying these changes associated with scleral
remodelling but are suitable only for ex vivo samples. Currently, no tool
is commercially available to inspect posterior scleral collagen in vivo.
On the basis of knowledge on scleral remodelling in myopic eyes, we
envision that a tool enabling in vivo imaging of collagen in the posterior
sclera could enable evaluation of the status of myopia, prediction of
its progression, identification of scleral weakening and prospective
evaluation of the risk of pathological changes.
Polarization-sensitive OCT (PS-OCT) derives image contrast from
tissue birefringence
33,34
and has been demonstrated to be a promising
tool for scleral collagen imaging in small animals in vivo35,36. Collagen
fibres exhibit a combination of form and intrinsic birefringence and
confer birefringence to scleral tissue, whereby light polarized along or
orthogonal to the fibre direction experiences slightly different refrac-
tive indices. Unlike the anterior sclera that can be directly accessed37,
imaging the posterior sclera in humans is far more challenging and
requires high detection sensitivity and accuracy, because the probing
light is attenuated
38
and the input polarization state is altered when
passing through the eye
39
. Recently, posterior sclera imaging using
PS-OCT has been shown in seven healthy volunteers and the archi-
tecture of scleral collagen fibres in normal eyes has been revealed
40
.
However, the clinical value of scleral collagen imaging is still unclear
and further investigations of scleral collagen imaging in preclinical and
clinical settings would benefit from further improvements in detection
sensitivity and system robustness.
In this study, we investigated posterior scleral birefringence (PSB)
in an animal model and in patients with myopia or pathologic myopia
using triple-input PS-OCT (TRIPS-OCT), a modulation and reconstruc-
tion strategy for PS-OCT that increases imaging sensitivity, accuracy
and system robustness. Current electro-optic modulator (EOM)-based
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Article https://doi.org/10.1038/s41551-023-01062-w
(32-yr-old male, oculus dexter (OD), Asian) (Fig. 1c). The in-plane ori-
entation of the HFL is approximately radially distributed around the
fovea52. We used the orientation of the HFL to assess the accuracy
of the optic axis measurements. We extracted the two-dimensional
corneal retardance and diattenuation maps from the surface of the
retina (Supplementary Method 6). In the optic axis images of the HFL,
the measured orientations were plotted against the angular location
on a circle centred on the foveal pit (Fig. 1d). An offset existed in the
measured optic axis orientation due to the unmeasurable circular bire-
fringence of the system and the anterior segment of the eye. The offset
can be estimated by minimizing the difference between the measured
optic axis orientation and the assumed orientation of the HFL, that is,
radial around the fovea. Without corneal correction, the measured fibre
orientation deviated markedly from the assumed radial profile. After
applying the correction for corneal retardance and diattenuation, the
mean error of the measurement, characterized by the residual differ-
ence between the measured optic axis orientation and the assumed
orientation (Fig. 1e), was 35% lower than that of the uncorrected results.
Scleral collagen architecture under TRIPS-OCT
To demonstrate imaging of the posterior scleral birefringence
(PSB), we scanned the eye of a healthy volunteer (28-yr-old female,
oculus sinister (OS), −6.75 dioptre (D), Caucasian) with TRIPS-OCT
in a three-dimensional volume and reconstructed the birefringence
and optic axis images (Supplementary Video 1). The cross-sectional
intensity image (Fig. 2a) obscured the complex scleral collagen fibre
structure, which was clearly visualized in the cross-sectional optic axis
image (Fig. 2b). Consistent with previous reports
40,53
, en face optic axis
images (Fig. 2c) of the peripapillary sclera showed a two-layer architec-
ture, where the inner and outer layers were dominated by radial and
circumferential fibres, respectively.
We used the eye of a pig, which was similar in size to a human eye,
to further validate TRIPS-OCT against polarized light microscopy
(PLM), an established tool for birefringence imaging. We first imaged
the pig’s eye in vivo with TRIPS-OCT and collected the eye for PLM his-
tological analysis immediately after imaging and killing the animal. The
TRIPS-OCT volume scan was rotated and resliced to register to the PLM
images. The radial and circumferential fibre distributions in the inner
and outer sclera layers indicated close agreement between TRIPS-OCT
(Fig. 2d,f) and PLM (Fig. 2e,g). The good co-location between the two
methods in the locations of fine structures, including the annular col-
lagen around the optic nerve head (ONH) and the tree-like stalk (Fig. 2h)
of the pig lamina cribrosa53, further validated the accurate registration
between these in vivo and ex vivo imaging modalities.
a
0
1.5
0 090 0.15
Fovea
c
d
Corneal retardance
(°)
Corneal diattenuation
(a.u.)
Not correct for corneal
birefringence
Correct for retardance Correct for retardance
and diattenuation
Correct for retardance
Not correct for corneal birefringence
Correct for retardance and diattenuation
Birefringence
(° µm–1)
0
90
Noise (° µm–1)
TRIPS
Dual-input
b
s.d. = 0.0550
s.d. = 0.0268
0.15
0.10
0.05
0
0 0.1 0.2
Probability
0 100 200 300
0
20
0
–20
0 100 200 300
100
200
300
Angular location around
the fovea (°)
Measured fibre
orientation (°)
Residual
error (°)
e
Orientation (°)
Fig. 1 | Technical advantages of TRIPS-OCT. a, TRIPS and dual-input
reconstruction methods on guinea pig retina in vivo. The intensity image (upper
left) and corresponding birefringence images are reconstructed from the
dual-input method (lower left) and the proposed triple-input method (lower
right). White boxes indicate the location of the zoomed-in views (upper right).
The reddish stripes in the inner retina that are present in the dual-input
reconstruction are induced by edge artefacts (Extended Data Fig. 3a,b). Of note,
the artefacts disappear in the TRIPS reconstruction. The orange area indicates a
region in the inner retina that is used to characterize the birefringence noise.
b, Histograms of birefringence noise calculated from the region in a indicated
by the orange area (pixel number n = 5,117 from 1 cross-sectional image). c,
Two-dimensional correction of corneal retardance and diattenuation. The en
face intensity image (upper left) from a healthy human subject (32-yr-old male,
OD, Asian) is rendered from a volume scan of the posterior eye. The
corresponding corneal retardance (upper middle) and diattenuation (upper
right) maps are extracted from the retinal surface and the optic axis images are
reconstructed without (lower left) and with the correction for corneal retardance
(lower middle), and for both retardance and diattenuation (lower right). The
position of the fovea is indicated by a white arrow. The magnitude of
diattenuation Dia is defined as the relative difference between the maximum
p2
1
and minimum
p2
2
attenuation coefficients, where
Dia =(p2
1−p2
2)/(p2
1+p2
2)
.
Zoomed-in images (right) indicated by white boxes highlight the HFL. d,
Measured in-plane HFL fibre orientation against angular location on a circle
(indicated by white dotted circles in c) centred on the fovea with an eccentricity
of 2°. e, Optic axis measurement error without/with corneal correction.
Scale bars: a, vertical: 300 µm, horizontal: 1 mm; c, 1 mm.
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ex vivo
in vivo ex vivo
in vivo
Retinal nerve fibre layer
Henle’s fibre layer
Inner sclera
Outer sclera
ab
d e f gc
ONH
Lamina fuscaInner scleraOuter sclera
Choroid
Inner sclera Outer sclera
Area
percentage (%)
Fibre orientation under TRIPS-OCT (°)
Percentage (%)
ij
k
j’
k’
l
m
n
o
Perpendicular
Oblique
Longitudinal
Perpendicular
Oblique
Longitudinal
Cells and other
1-week-old 12-week-old
1.5
0.02
1-week-old
12-week-old
qp
1-week-old12-week-old
r
h
Birefringence (° µm–1)
Percentage (%)Percentage (%)Percentage (%)
Collagen fibre diameter under TEM (µm)
Birefringence under TRIPS-OCT (° µm–1)
Collagen fibre diameter (µm)
Inner sclera
Outer sclera
Inner sclera
Outer sclera
Inner sclera Outer sclera
Outer
Inner
0
30
60
20
10
0
20
10
0
00.05 0.10 0.1 5 0.20 0.25
050 100
20
10
000.2 0.4 0.6 0.8
0
0
10
20
0.100.05 0.15 0. 20 0.25
Fig. 2 | Validation and interpretation of TRIPS-OCT images. a–c, TRIPS-OCT
scan on a healthy participant (28-yr-old female, −6.75 D, Caucasian) showing a
representative cross-sectional intensity image (a) and the corresponding optic
axis image (b), and the en face optic axis images (c) centred at the ONH at two
depths. d–g, Pig en face scleral optic axis images at two depths under TRIPS-OCT
in vivo (d,f) and registered images under PLM ex vivo (e,g). White arrows in d and
e indicate the annular collagen around the ONH. White boxes in f and g indicate
the tree-like stalk of the pig lamina cribrosa. Eyeball deformation during tissue
fixation creates discrepancy areas in d and e indicated by white dashed lines.
h, Magnified views of dashed boxes in f and g. i–k, Guinea pig TEM images of the
sclera (i), the inner (j) and outer (k) sclera and zoomed-in views to observe the
fibres. Perpendicular, longitudinal and oblique fibre orientations are colour-
coded in j’ and k’. l,m, Percentages of the fibre orientations (l) and diameter
(m) measured from TEM images. Each histogram is calculated from n = 600
individual fibres from 1 sclera sample. n,o, Distributions of fibre orientation (n)
and birefringence (o) measured from TRIPS-OCT in vivo (Supplementary Data
Fig. 1) at roughly the same location as TEM. Each histogram is calculated from
n = 500 pixels in a cuboidal region from 1 volume scan. p, Guinea pig en face
images in one eye at the ages of 1 week and 12 weeks. q, TEM images at the ages
of 1 week and 12 weeks. r, Outer scleral collagen fibre diameter distributions
measured from TEM images from 2 guinea pigs at the ages of 1 week and 12
weeks. Histogram equalization was applied to a. Scale bars: a, vertical: 300 µm,
horizontal: 1 mm; c,d,f,p, 1 mm; h, 150 µm; i, 30 µm; k,k', 10 µm; q, 2 µm.
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Article https://doi.org/10.1038/s41551-023-01062-w
Birefringence reflects the arrangement and diameter of fibres
To further interpret the birefringence images, we compared TRIPS-OCT
with transmission electron microscopy (TEM), a nanometre-scale reso-
lution tool that can clearly visualize collagen bundles and individual
fibres. A 16-week-old guinea pig’s eye was imaged in vivo via TRIPS-OCT
(Supplementary Data Fig. 1) and then collected for TEM analysis after
killing the animal. The TEM section was sampled parallel to the tem-
poral–nasal plane in the superior region located 2 mm from the ONH.
From the TEM images (Fig. 2i–k) of the inner and outer sclera, the fibre
orientations with respect to the sectioning plane were classified as
perpendicular, longitudinal and oblique (Fig. 2j’,k’). We calculated the
percentages of the different fibre orientations (Fig. 2l) and measured
the fibre diameter distribution (Fig. 2m). For comparison, in roughly the
same location, we evaluated the distributions of optic axis orientation
(Fig. 2n) and birefringence (Fig. 2o) measured with TRIPS-OCT in vivo.
We qualitatively observed that the measured optic axis orientations cor-
responded to the average orientation of all fibres within the TRIPS-OCT
resolution volume. By inspecting the birefringence images, we found
that higher birefringence corresponded to more aligned fibres within
the TRIPS-OCT resolution volume, explaining the ring-like pattern
around the ONH in the en face birefringence image (Fig. 2p), where
most of the fibres were circumferentially arranged around the ONH.
To qualitatively investigate the relationship between birefringence
and collagen fibre diameter, we imaged the same eye of one guinea
pig in vivo at the ages of 1 week and 12 weeks using TRIPS-OCT and
reconstructed the en face birefringence images (Fig. 2p). A significant
increase in scleral birefringence was observed in the older animal due
to physiological eye growth. We then killed two guinea pigs at the ages
of 1 week and 12 weeks for TEM analysis. TEM sectioning (Fig. 2q) was
performed at the outer sclera from the same location in both animals
and the distributions of scleral fibre diameters (Fig. 2r) were calculated.
We observed that in the older animal, the distribution of fibre diameter
was skewed to larger values. Overall, in addition to more aligned fibres,
a larger average collagen fibre diameter, corresponding to a higher
collagen content and thicker lamellae (Supplementary Discussion 2),
led to the increased birefringence in TRIPS-OCT images.
Development of refractive errors in guinea pigs
To explore the correlation between posterior scleral birefringence (PSB)
and the development of refractive errors, we used a cohort of guinea
pigs (N = 21), mixed albino (N = 17) and pigmented (N = 4) strains, which
were previously reported to have spontaneous myopia rates of 70%
(albino) and 29% (pigmented)
47
. We imaged the animals weekly with
TRIPS-OCT from birth to 8 weeks (Fig. 3a, raw data in Supplementary
Figs. 2 and 3). As the optical aberration of the eye at the age of 1 week
hindered TRIPS-OCT imaging, some measurements were not available
at the age of 1 week. Refraction was measured as spherical equivalent
refractive error (SE) with retinoscopy from 1 to 8 weeks. Notably, we
observed strong correlations between SE and PSB from the ages of 2
to 8 weeks (Fig. 3b), with the highest Pearson correlation coefficient
observed at the age of 4 weeks (r = 0.78, P = 3.6 × 10−5).
We also performed a repeatability test of the birefringence meas-
urements (Extended Data Fig. 4) within a cross-sectional imaging exper-
iment of guinea pig eyes in vivo. Excellent measurement repeatability
of PSB was demonstrated between repeat measurements at the same
imaging angle (r = 0.999, 1.96 s.d. = 2.43%, 12 eyes) and between meas-
urements at two different imaging angles (r = 0.995, 1.96 s.d. = 6.09%,
12 eyes).
PSB predicts the onset of myopia in guinea pigs
To evaluate the PSB measured at the age of 2 weeks and myopia onset
at the age of 4 weeks, we used the data obtained from the longitudinal
model described in the previous subsection. TRIPS-OCT images meas-
ured at the age of 2 weeks were assigned to two groups (Fig. 4a,b) on the
basis of the SE measurements at the age of 4 weeks by a threshold of 0 D
(myopia group: SE < 0D, emmetropia and hyperopia group: SE ≥ 0D).
We observed that guinea pig eyes in the myopia group showed signifi-
cantly lower PSB than those in the emmetropia and hyperopia group
(P = 0.0054, Fig. 4c).
We hypothesized that PSB could be a predictor for the onset of
myopia and compared it to baseline SE, which has been reported as the
best single predictor for myopia onset
48,49
. We assessed the correlation
between the baseline SE, PSB measured at the age of 2 weeks and the
SE at the subsequent ages of 2–8 weeks (Fig. 4d). Notably, from the age
of 4 weeks onwards, the baseline SE was less correlated with refraction
status than the PSB.
We next used the baseline SE and PSB at the age of 2 weeks to
predict myopia onset (defined as SE < 0D) at the ages of 4 and 8 weeks.
Receiver operating characteristic (ROC) curves (Fig. 4e) of prediction
outcomes showed that PSB achieved better performance than the
baseline SE (week 4: PSB area under the curve (AUC), 0.89; baseline SE
AUC, 0.74; week 8: PSB AUC, 0.85; baseline SE AUC, 0.73).
Birefringence is correlated with myopia status in humans
To investigate whether a correlation between PSB and myopia status
exists in humans, 80 participants without pathological ocular condi-
tions were recruited (Extended Data Fig. 5). TRIPS-OCT scanning and
measurements of SE and axial length (AL) were performed on both eyes
of every participant. Due to the requirement for a sufficient signal from
the sclera, we excluded 75 eyes (47%) on the basis of quality criteria
(Supplementary Discussion 3), composed of images with suboptimal
positioning (50 eyes, 31%) and insufficient signal-to-noise ratio (aver-
age scleral SNR < 4.6 dB) from the sclera (25 eyes, 16%). In TRIPS-OCT
images (Fig. 5a–h and Supplementary Video 2) of a typical emmetropic
eye (SE = 0 D, Fig. 5a,c,e) and a myopic one (SE = −6.75 D, Fig. 5b,d,f–h),
we observed that the myopic eye presented increased PSB, both in the
outer peripapillary area and the posterior pole area.
We then investigated the correlation between PSB and myopia
status in participants with emmetropia or low degree of myopia. Using
SE as a threshold, we grouped the eyes into two groups: the emmetro-
pia or low myopia group (−6D < SE ≤ 3D) and the high myopia group
(SE ≤ −6D). From the 69 eyes of 42 participants (Supplementary Table 1)
in the emmetropia or low myopia group, we calculated the scleral bire-
fringence at two different locations on the fundus; these values were
the outer peripapillary scleral birefringence (OPSB) and the posterior
pole scleral birefringence (PPSB) (Fig. 5e). We assessed the correlation
between SE and both OPSB and PPSB (Fig. 5i,j) and found that SE was
significantly correlated with PPSB and to a lesser degree with OPSB
(PPSB vs SE: r = −0.60, P = 1.2 × 10
−4
, OPSB vs SE: r = −0.42, P = 0.012).
We further assessed the correlation between PPSB and AL (Fig. 5k) to
confirm the strong correlation between myopia status and PPSB (PPSB
vs AL: r = 0.54, P = 0.001).
To further investigate the location dependence of PSB and myopia
status on the fundus, the outer peripapillary area of the eyes from the
emmetropia or low myopia group was divided into 12 segments by
polar coordinates (Fig. 5e) and the mean birefringence value within
each segment was correlated with the SE and AL (Fig. 5l and Supple-
mentary Data Fig. 4). Notably, the birefringence in the 4 segments
close to the fovea exhibited a significant correlation with myopia status
(temporal-inferior: −60°, −30°; temporal: 0°; temporal-superior: 30°;
P ≤ 0.01) and the highest Pearson correlation coefficient was observed
in the segment located between the ONH and the fovea (temporal: 0°),
which served as the definition of PPSB.
In addition, to evaluate the correlation among other biometrics
within the eyes in the emmetropia or low myopia group, we addition-
ally measured the choroidal thickness and the inner sclera thickness
from the TRIPS-OCT images. We evaluated the correlation among age,
AL, SE, choroidal thickness, inner scleral thickness and PPSB (Fig. 5m;
model parameters in Extended Data Table 1, raw data in Supplementary
Data Fig. 5). We found that in addition to the known strong correlation
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between SE and AL, the correlation between PPSB and myopia status
(SE, AL) was significantly higher than that between other biometrics.
PSB is associated with pathological changes in humans
We hypothesized that PSB would be an indicator of scleral pathological
changes. To test this hypothesis, we recruited 10 patients with both
eyes diagnosed with myopia-associated staphyloma. TRIPS-OCT and
AL measurements were performed on both eyes, but no SE measure-
ments were performed due to the low function of the eyes. Fifteen eyes
(75%) were scanned and included in the pathologic myopia group for
further analysis (Supplementary Table 1).
In eyes with pathologic myopia, we observed a spatial asso-
ciation between PSB and staphyloma. From the TRIPS-OCT images
(Fig. 6a–c) of a typical eye with staphyloma, we further reconstructed
the three-dimensional eye shape (Fig. 6d and Supplementary
Video 3) from the volume scan and identified the edges of staphy-
loma. We observed that increased PSB was spatially correlated with
the staphylomatous outpouching regions. To observe the PSB in eyes
with various stages of myopia, we compared the eyes from the high
myopia group reported in the previous subsection and the eyes in the
pathologic group (Fig. 6e). We observed that PPSB markedly increased
in eyes with pathologic myopia even when staphyloma edges were not
located in the posterior pole area.
To further investigate the correlation between PPSB and myo-
pia status, we combined the eyes from the emmetropia or low myo-
pia group, the high myopia group and the pathologic myopia group
and assessed the correlation between PPSB and AL (Fig. 6f). A strong
correlation was found between PPSB and AL in these eyes (r = 0.55,
P = 3.9 × 10
−5
, 100 eyes), verifying that the increase in PPSB is associated
with scleral changes in pathologic myopia.
To further evaluate the potential of PPSB as a marker to differenti-
ate eyes with pathologic changes, we combined the eyes from the high
myopia group and the pathologic myopia group and compared PPSB to
AL as classifiers to identify pathological eyes (Fig. 6g). The PPSB showed
better performance than the AL in terms of the AUC (PPSB AUC: 0.94,
95% CI (0.72, 1); AL AUC: 0.82, 95% CI (0.53, 1)).
To evaluate whether PPSB indicates potential progression of high
myopia, we further divided the high myopia group according to the
presence of peripapillary atrophy (PPA), which has been reported as a
factor associated with progressive myopia
50,51
. We observed that PPSB
was significantly higher in eyes with PPA than in those without PPA
(P = 0.011, Fig. 6h). The PPSB of eyes in the pathologic myopia group
was higher than that of eyes in the high myopia group, albeit with sta-
tistical significance only for eyes without PPA (P = 9.1 × 10
−5
vs P = 0.18,
Fig. 6h). Consistent with the diagnostic implication of PPA, increased
PPSB may prospectively suggest the potential for progression from
high myopia to pathologic myopia.
Discussion
The prevalence of myopia is increasing globally. It has been predicted
that myopia will affect almost 5 billion people by 2050. Although only a
fraction thereof would develop pathologic myopia, this will amount to
approximately 300 million people
1,2
. Patients with pathologic myopia
have reduced quality of life due to its economic and societal impact54.
Myopia is now recognized as an immense future healthcare problem
that needs to be addressed today
55
. While genetic, environmental, bio-
chemical and physiological factors have been reported to contribute to
the development and progression of myopia1, the physical elongation of
the eye is ultimately related to the remodelling of the posterior sclera56.
In this study, we measured PSB, which relates to the architecture and
diameter of collagen fibres in the posterior sclera, and investigated its
potential as a biomarker for predictively evaluating the risk of myo-
pia progression. To enable clinical PSB measurements, we developed
TRIPS-OCT which offers benefits for birefringence imaging in terms
Guinea pig 1 ODGuinea pig 2 OD
a
r = 0.39
P = 0.079
r = 0.68
P < 0.001
r = 0.78
P < 0.001
r = 0.68
P < 0.001
r = 0.63
P = 0.0021
r = 0.63
P = 0.0027
r = 0.48
P = 0.027
2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks
Spherical equivalent refractive error (D)
Posterior scleral
birefringence (°/µm)
b
OS
OD
1.50.02
Age:
Age: 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks
Birefringence (° µm–1)
–5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
0.4
0.6
0.8
1.0
Fig. 3 | Correlation between the development of refractive errors and
posterior scleral birefringence as measured using TRIPS-OCT in a guinea
pig model. a, Representative guinea pig en face scleral birefringence images of
two eyes longitudinally measured weekly from 1 week to 8 weeks of age.
Scale bar, 1 mm. b, Correlation analysis of posterior scleral birefringence
measured from the images and SE from 2 weeks to 8 weeks of age. Scatterplots
show 42 individual eyes from 21 animals, regression (lines) and 95% confidence
intervals (shaded areas). r values were calculated using Pearson correlation.
P values were calculated using F-test against a constant model. Inter-eye
correlation was addressed by cluster bootstrapping.
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of sensitivity, accuracy, robustness and imaging range, compared with
previous PS-OCT implementations.
In our guinea pig model, we showed that the development of
refractive errors from the ages of 2–8 weeks was correlated with PSB.
Myopia onset at the ages of 4 and 8 weeks can be predicted by PSB
measured at 2 weeks. Various studies have demonstrated that the
establishment of refraction is controlled through the modulation of
scleral extracellular matrix growth and remodelling57. Specifically, in
response to myopiagenic visual signals (that is, defocus), the activity
of scleral fibroblasts, chondrocytes and myofibroblasts (regulated by
gene expression or biochemical factors) is altered, sequentially altering
extracellular matrix synthesis and organization
19,58
. The PSB measured
at the age of 2 weeks is related to the average collagen fibre diameter
in the posterior sclera. As a possible explanation of our results, PSB is
indicative of the level of scleral collagen synthesis19 and relatedly, the
proliferation of myofibroblasts
58
. Therefore, the rapid increase in PSB
in young animals during eye growth may indicate the establishment
of an effective regulation from vision to scleral development18, which
reduces the animals’ susceptibility to myopia.
To control early-stage myopia, increasing outdoor activities can
be unequivocally encouraged for all children. However, more targeted
interventions, such as orthokeratology and atropine eye drops, have
to be applied selectively on the basis of the individual risk of develop-
ing high myopia8,13,59. The prediction of myopia progression is par-
ticularly important for guiding treatment decisions. Various studies
have reported that baseline SE is the best single predictor for myopia
onset48,49, outperforming other risk factors, including outdoor time and
parental myopia. In contrast, in our guinea pig model, we demonstrated
that PSB performed better than baseline SE with higher AUC values. Our
results imply that PSB may serve as a predictive biomarker for predict-
ing myopia progression in children; however, further clinical studies
focusing on children participants are needed to validate the associa-
tion between PSB and childhood myopia. Considering active research
into various myopia control strategies, such as atropine with different
0
0.5
1.0
0 0.5 1.0
0 0.5 1.0
0
0.5
1.0
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
–5
0
5
10
0.6 0.8 0.6 0.8 0.6 0.8 0.6 0.8 0.6 0.8 0.6 0.8 0.6 0.8
–5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10 –5 0 5 10
–5
0
5
10
1-specificity
Sensitivity
Predict myopia onset at 4 weeks
of age from data collected at 2
weeks of age
Predict myopia onset at 8 weeks
of age from data collected at 2
weeks of age
a
d
1.50.02
Birefringence at week 2 (° µm–1)
P = 0.0054
0.8
0.7
0.6
0.5
Myopia group
Emmetropia and
hyperopia group
c
Baseline spherical equivalent refractive error (D)
Posterior scleral birefringence at the age of 2 weeks (° µm–1)
3 weeksAge: 2 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks
r = 1
P = 0
r = 0.73
P < 0.001
r = 0.55
P = 0.010
r = 0.53
P = 0.013
r = 0.42
P = 0.059
r = 0.27
P = 0.22
r = 0.30
P = 0.18
r = 0.39
P = 0.095
r = 0.67
P < 0.001
r = 0.76
P < 0.001
r = 0.65
P = 0.0013
r = 0.65
P = 0.0013
r = 0.69
P < 0.001
r = 0.63
P = 0.0024
PSB
AUC = 0.89
Baseline SE
AUC = 0.74
Sensitivity
PSB
AUC = 0.85
Baseline SE
AUC = 0.73
e
OS
OD
Spherical equivalent refractive error (D)
Birefringence (° µm–1)
b
Fig. 4 | Prediction of myopia onset using TRIPS-OCT in guinea pig model.
a,b, En face scleral birefringence images measured at the age of 2 weeks from the
entire cohort of guinea pigs. Images are grouped by myopia outcome at the age
of 4 weeks, defined as SE < 0D. Group 1 (a), myopic eyes. Group 2 (b), emmetropic
and hyperopic eyes. Scale bar, 1 mm. c, PSB values measured from the images
in the two groups of guinea pig eyes. Dots represent n = 42 eyes from 21 guinea
pigs, central line indicates median, box shows interquartile range and whiskers
show range. P value was calculated using two-sided Wilcoxon rank-sum test with
cluster bootstrapping to correct inter-eye correlation. d, Correlation between
the predictors, baseline SE (upper panel) and PSB at the age of 2 weeks
(lower panel), and the outcome (SE at the ages of 2–8 weeks). Scatterplots
show 42 individual eyes from 21 animals, regression (lines) and 95% confidence
intervals (shaded areas). r values were calculated using Pearson correlation.
P values were calculated using F-test against a constant model. Inter-eye
correlation was addressed by cluster bootstrapping. e, Predictions of myopia
outcomes at the ages of 4 weeks (upper panel) and 8 weeks (lower panel) from
the data measured at the age of 2 weeks, using PSB (orange line) and baseline SE
(blue line) as predictors. PSB: AUC, 0.89; 95% CI (0.70, 1); SE: AUC, 0.74, 95% CI
(0.48, 0.94); Week 8, PSB: AUC, 0.85, 95% CI (0.61, 1); Baseline SE: AUC, 0.73, 95%
CI (0.46, 0.95).
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P = 0.01
P = 0.001
P < 0.001
P < 0.001
P < 0.001
22 24 26 28 –150 –90 –30 30 90 150
0.2
0.4
0.6
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–6 –4 –2 0 2 4
0.2
0.4
0.6
Spherical equivalent
refractive error (D)
Outer peripapillary scleral
birefringence (° µm
–1
)
Posterior pole scleral
birefringence (° µm
–1
)
Posterior pole scleral
birefringence (° µm
–1
)
r = –0.26
P = 0.112
r = 0.11
P = 0.433
r = –0.03
P = 0.398
r = 0.44
P = 0.007
r = –0.33
P = 0.057
r = 0.54
P = 0.001
r = –0.23
P = 0.168
r = –0.01
P = 0.365
r = –0.69
P < 0.001
r = –0.60
P < 0.001
r = 0.17
P = 0.286
r = 0.11
P = 0.424
r = –0.27
P = 0.113
r = –0.17
P = 0.281
r = 0.17
P = 0.279
0°
–150°
30°
–120°
60°
90°
120°
150°
180°
–90° –60°
–30°
Fovea
n = 69
r = –0.42
P = 0.012
T
N
I
S
Axial length (mm)
a
a’
a”
b
b’
b”
cd
c’ d’
e
ff’
e’
gh
i j
m
n = 69
r = –0.60
P < 0.001
OS
OD
n = 69
r = 0.54
P = 0.001
Location (°)
Pearson correlation
coeicient r
kl
Spherical equivalent
refractive error (D)
OPSB
PPSB
1
0
Pearson correlation coeicient r
PPSB vs SE
PPSB vs AL
Posterior pole
scleral
birefringence
Inner scleral
thickness
Choroidal
thickness
Refractive
error
Axial
length
Age
Posterior pole scleral
birefringence
Inner scleral thickness
Choroidal thickness
Refractive error
Axial length
Age
****
****
P = 0.001
P = 0.002
P = 0.007
–6 –4 –2 0 2 4
0.2
0.4
0.6
1.50.05
Birefringence
(° m
–1
)
Fig. 5 | Scleral birefringence in non-pathologic patients with myopia.
a–f, Representative TRIPS-OCT images of an emmetropic eye (40-yr-old female,
OS, 0 D, Caucasian) (a,a’, a”,c, c’,e,e’) and a myopic eye (28-yr-old female, OS,
−6.75 D, Caucasian) (b,b’,b”,d,d’,f,f’). Cross-sectional images containing the
ONH (a,b), fovea (c,d) and en face images (e,f) are shown in intensity (a–d,e,f),
birefringence (a’–d’,e’,f’) and optic axis (a”,b”) contrasts. Dotted lines in e and
f indicate locations of cross-sectional images. g, Zoomed-in view of the box in
f’ and corresponding optic axis image showing the circumferential ring-like
structure. h, Cross-sectional intensity (left) and birefringence (right) images
indicated by the white dashed line in f’ showing the uneven choroidal–scleral
interface (orange dashed line) resulting in the petaloid birefringence pattern
(black arrowheads in f’ and h). En face birefringence and optic axis images were
obtained from an average projection of a 200 µm slab centred at the manually
labelled choroidal–scleral interface (blue dashed line). Scleral birefringence
values were calculated by averaging specific fundus areas indicated by orange
annular (OPSB) and hatched segment (PPSB) in e. i–k, Correlation analysis
of OPSB vs SE (i), PPSB vs SE (j) and PPSB vs AL (k). l, Pearson correlation
coefficients of PPSB vs SE and PPSB vs AL at different fundus locations. *P ≤ 0.01.
m, Correlation matrix of biometrics of the eyes in the emmetropia or low myopia
group. Scatterplots (i,j,k) show 69 eyes from 42 individuals, regression (lines)
and 95% confidence intervals (shaded areas). r values were calculated using
Pearson correlation. P values were calculated using F-test against a constant
model. Inter-eye correlation was addressed by cluster bootstrapping. Histogram
equalization was applied to a, b, c, d and h (left panel). S, superior; I, inferior;
N, nasal; T, temporal. Scale bars: a,a”,c, vertical: 300 µm, horizontal: 1 mm;
e, 1 mm.
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0
0.2
0.4
0.6
0.8
1.0
0
High myopia w/o PPA
High myopia w PPA
Pathologic myopia
0.2
0.4
0.6
0.8
1.0
0 0.5 1.022 24 26 28 30 32
0
0.2
0.4
0.6
0.8
1
2
PPA
High myopia Staphyloma
e
aa’ b
b’ c’
c
0.05
d
Posterior pole scleral
birefringence (° µm–1)
Axial length (mm) 1-specificity
Sensitivity
Emmetropia or low myopia (>–6D)
High myopia (≤−6D)
Pathologic myopia with staphyloma
Eyes with PPA
Posterior pole scleral
birefringence (° µm–1)
n = 100
r= 0.55
P < 0.001
Non-pathologic vs pathologic
Ground truth: eye shape (OCT)
PPSB
AUC = 0.94
95%CI: 0.72, 1
Axial length
AUC = 0.82
95%CI: 0.53, 1
P = 0.011
P = 0.18
P < 0.001
f g h
0.05 1.50
Birefringence (° µm–1)
Birefringence (° µm–1)
Fig. 6 | Scleral birefringence in patients with high and pathologic myopia.
a–d, Representative TRIPS-OCT images of a patient (61-yr-old female, OD,
AL: 27.8 mm, Asian) with pathologic myopia, diagnosed by the presence of
staphyloma, shown in contrasts of en face intensity (a), en face birefringence
(a’), cross-sectional intensity (b,c), cross-sectional birefringence (b’,c’) and
three-dimensional reconstruction of the eyeball shape (d). The dashed lines in
a and a’ indicate the locations of the cross-sectional images (b,b’,c,c’). White
arrowheads (a’,b,b’,c,c’) indicate the edges of staphyloma. Yellow arrowheads
(b’,c’) indicate regions of scleral birefringence increase. e, Representative images
of eyes with high myopia and pathologic myopia at various stages (from left to
right: 32-yr-old male, OD, AL: 28.8 mm; 32-yr-old female, OS, AL: 26.9 mm; 49-yr-
old female, OS, AL: 26.9 mm; 62-yr-old male, OS, AL: 30.2 mm, all Asian). Images
are shown in contrasts of cross-sectional intensity (upper panel), en face intensity
(middle panel) and en face birefringence (lower panel). Black arrows indicate
PPA. f, Correlation of PPSB and AL in all eyes (non-pathologic n = 85, pathologic
n = 15). The scatterplot shows 100 eyes from 59 individuals, regression (line) and
95% confidence intervals (shaded area). The r value was calculated using Pearson
correlation. The P value was calculated using F-test against a constant model.
Inter-eye correlation was addressed by cluster bootstrapping. g, Performance
of using PPSB and AL as classifiers to differentiate eyes with pathologic myopia
from eyes with high myopia. h, PPSB values in eyes with high myopia without
PPA (n = 10), high myopia with PPA (n = 6) and pathologic myopia (n = 15). Dots
represent eyes, central line indicates median, box shows interquartile range and
whiskers show range. The P value was calculated using two-sided Wilcoxon rank-
sum test with cluster bootstrapping to correct inter-eye correlation. Histogram
equalization was applied to b, c and e (upper panels). Scale bars: a, 1 mm;
b,e, vertical: 300 µm, horizontal: 1 mm.
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doses and combinative treatment of atropine and orthokeratology60,
PSB could potentially complement the widely used SE for optimizing
the treatment strategy and tailoring it to individual needs.
In our human cross-sectional study, we showed that in eyes with
emmetropia and low myopia, PPSB increased on average by 0.03° µm
−1
as myopia progressed by −1 D, and increased by 0.05° µm−1 as AL
increased by 1 mm. Furthermore, in patients with pathologic myopia,
we observed a spatial association between PSB and staphyloma in the
sclera. In eyes with high myopia, we found that PSB increased when
PPA was present and might predict further progression of the disease.
In the posterior pole area, we observed the most pronounced cor-
relation of PSB with SE and AL. This observation is in agreement with
previous observations in animals, which identified that the primary
myopic change in the sclera started from the posterior pole area61,62.
The increase in PSB in myopic eyes can be explained by changes in the
collagen fibre structure from interwoven to aligned, as observed in ex
vivo studies, including (1) unfolding of microstructural crimps
22,29
, (2) a
change in collagen fibre direction from circumferential to radial in the
peripapillary sclera
30
, (3) reorganization of the collagen fibres into a
lamellar arrangement (rather than interwoven)
63
or (4) a combination
of all aforementioned phenomena21.
In the clinical management of pathologic myopia, PSR surgery
(for example, macular buckling) has been an option to arrest myopia
progression. In general, due to its invasive nature and concerns about
post-operative complications, PSR is only considered when ocular
pathologies such as retinal detachment and myopic maculopathy
(caused by progressive thinning of the sclera) are threatening or already
impacting vision
60
. There is a need to detect scleral weakness as early as
possible and for close monitoring to make a timely treatment decision
7
.
Our results in humans showed that PSB was correlated with myopia
degree and may be a sensitive indicator of scleral degeneration towards
pathological conditions. TRIPS-OCT is sensitive to scleral changes
in moderate and low myopia, which are not generally observed with
current fundus photography or conventional OCT
64
. We speculate
that TRIPS-OCT can detect subtle changes in the sclera that precede
an obvious change in thickness or morphology accessible with current
imaging methods, and can thus provide better guidance on the neces-
sity and timing of PSR treatment.
Comparing the correlations between PSB and the degree of myo-
pia in the young guinea pigs and adult humans, the PSB decreased in
animals but increased in adult humans with the severity of the disease.
We additionally imaged two adult guinea pigs without and with high
myopia (Extended Data Fig. 6) and found the PSB was higher in the
myopic eye, which is consistent with the data obtained in humans. We
suppose that there is a fundamental difference in the scleral changes
between the young guinea pig model and adult humans represent-
ing different stages of myopia development within the lifespan. Our
data indicate that the early eye growth at younger age and the myopic
elongation in adults lead to opposing effects on PSB related to scleral
collagen fibre arrangement and diameter. To understand the differ-
ence between these two stages of myopia development, we analysed
the collagen fibre orientation and birefringence within the regions
of interest in guinea pigs and humans. In the young guinea pigs at
the ages of 2 and 8 weeks (Extended Data Fig. 7), the average scleral
birefringence increased with age, in conjunction with an increase in
the interweaving of the collagen fibres as evidenced by a reduction in
the local maxima in the angular histograms of fibre orientation. In the
adult guinea pigs, the average birefringence was higher in the myopic
eye, however, in conjunction with a reduction in the interweaving of
the collagen fibres (Extended Data Fig. 6). The latter phenomenon was
also observed in the submacular sclera of adult humans (Extended
Data Fig. 8). As such, increased PSB in young guinea pigs may indicate
the enlargement of scleral fibre diameter, associated with increased
scleral collagen synthesis to achieve the required scleral stiffness
during eye growth. In contrast, increased PSB in adult guinea pigs and
humans with myopia may indicate alterations in the arrangement of
scleral collagen and reductions in interwoven fibres, associated with
an extended elongation of the eyeball. However, these suppositions
are solely based on TRIPS-OCT measurements and still require further
histological investigations.
In this study, we introduced TRIPS-OCT, a new polarimetric modu-
lation and reconstruction strategy for posterior scleral imaging in vivo.
Today, there are three major types of PS-OCT instruments, including
single-input PS-OCT
65,66
, depth-encoding PS-OCT
45,46,67
and dual-input
EOM-based PS-OCT41,42. Single-input PS-OCT offers a simplified setup
but limits its use to lowly birefringent samples. When the polarization
state of the local probing light occasionally coincides with the sam-
ple’s optic axis, the depth-resolved reconstruction of birefringence
metrics may be frustrated. Depth-encoding PS-OCT offers reliable
depth-resolved measurements independent of the sample but requires
a doubled ranging depth to achieve the same imaging depth as a time
multiplexing system and a k-clock device to ensure phase stability
68
.
The dual-input EOM-based PS-OCT is robust to environmental variation
as it does not require phase stability for birefringence reconstruction,
but it assumes measurements to represent pure retardance, hence it is
readily impacted by the presence of diattenuation and edge artefacts
induced by both the system components and the sample. TRIPS-OCT
does not have the aforementioned limitations but requires a longer
acquisition time because of triple repetitive scans at the same loca-
tion of the sample. However, due to the development of faster lasers
and OCT-angiography techniques
69
, repetitive scanning has become
a standard in current ophthalmic imaging. TRIPS-OCT is not sensitive
to sample motion within a few micrometres, as the measurements of
modulated frames are based on Stokes vectors, and the phase variation
between repetitive scans does not influence the reconstruction. Over-
all, TRIPS-OCT mitigates some of the drawbacks of traditional PS-OCT
implementations and makes it more suitable for clinical translation.
This study has several limitations that we hope can be addressed.
First, TRIPS-OCT measurements are fundamentally limited by the
intensity SNR. As the sclera is a dense and highly scattering structure,
only a 100-µm band from the upper sclera can be reliably measured. In
this study, we estimated the intensity SNR of the 100-µm scleral band
and excluded approximately 16% of all imaged eyes due to insufficient
intensity SNR. We found that a thick choroid (roughly >450 µm) was one
of the factors limiting light penetration to the sclera. In addition, we
observed that Caucasian eyes provide slightly better penetration of the
sclera than Asian eyes, perhaps due to less scattering and absorption
caused by lesser pigmentation. Second, as both the diameter and the
alignment of the collagen fibres determine the birefringence meas-
ured by TRIPS-OCT, the explanation of increased PSB requires further
analysis of the collagen fibre orientation and previous knowledge of
the underlying structures. Lastly, the scleral birefringence measure-
ment highly depends on the segmentation of the choroidal–scleral
interface, which is performed manually at present. The issue with
manual segmentation is that the interface between the choroid and
the sclera is not well defined. There are fine petaloid scleral structures
unevenly connecting to the above choroid tissues and large blood ves-
sels going through the sclera from the choroid. To minimize the trun-
cation of the inner parts of the sclera, we used a 200-µm band centred
on the choroidal–scleral interface to produce the en face images and
quantify birefringence. Incorporating an area including the choroid
does not affect the PSB measurement because no structure in the
choroid is observed to be birefringent. In addition, manual labelling is
a time-consuming and subjective process, whereas an automated and
reliable image segmentation algorithm will improve the accuracy and
efficiency of TRIPS-OCT analysis.
We have reported the development of a polarimetric imaging
technique, TRIPS-OCT, and revealed PSB as a biomarker for myopia
by imaging the posterior sclera in eyes with different presentations
of myopia. We anticipate TRIPS-OCT to be potentially useful in the
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diagnosis of other ocular conditions that are related to scleral anoma-
lies. Furthermore, TRIPS-OCT may be applied to imaging systems based
on fibre probes, thus bringing new opportunities in intravascular and
endoscopic applications.
Methods
Triple-input modulation
A swept-source optical coherence tomography (SS-OCT) system previ-
ously developed by our group70 was modified to achieve TRIPS-OCT.
The OCT system employed a swept-source laser (1,060 nm, sweep rate
200 kHz, tuning range 100 nm, Axsun Technologies). The measured
axial full-width-at-half-maximum of the intensity PSF in air was 6.1 µm.
In a swept cycle, the digitization was triggered by the laser trigger
signal with a constant sampling rate of 1 GHz, and the measured 3 dB
roll-off ranging depth was 3.5 mm. The beam size entering the pupil was
0.67 mm, corresponding to an optical lateral resolution of 44 µm and
a depth of focus of 2.9 mm in a normal human eye with an axial length
of 23 mm. The spatial averaging in the birefringence reconstruction,
including filtering of Stokes vectors and spectral binning, reduced the
resolution to 150 µm in the lateral and 30 µm in the axial directions for
birefringence imaging. The laser power entering the eye, which was
controlled by a motorized aperture placed in the free space before
the triple-input modulator, was set to 1 mW for alignment and 4 mW
for retinal volume scan.
We replaced the polarization-depth-encoding unit with a
triple-input modulator (Extended Data Fig. 1b) consisting of a polar-
izer and an EOM (4104NF, Newpor t), inspired by the previous dual-input
configuration
71
. A triple-step voltage driving signal (Extended Data
Fig. 1c) and an angle of 27.37° between the optic axis of the EOM and
its preceding linear polarizer (Extended Data Fig. 1d) allowed for the
generation of three mutually orthogonal polarization states on the
Poincaré sphere (Extended Data Fig. 1e and Supplementary Method 3).
Modulating the polarization states between three repeated frames
enabled the reconstruction of the Mueller matrix at each location
within the triple-measured frames. The reconstruction involved an
algorithm (Supplementary Method 1) fitting the measured Stokes
vectors to Mueller matrices that respect physical polarization con-
straints and describe cumulative diattenuation and retardance. From
the reconstructed Mueller matrices, we isolated the cumulative sample
retardance and computed local depth-resolved tissue birefringence
and optic axis orientation.
Mueller matrix reconstruction
To remove wavelength-dependent polarization effects, spectral bin-
ning was performed by dividing the spectral fringe into 9 overlap-
ping bins. The signals from the 2 detection channels were converted
to Stokes vectors and filtered along both fast and slow lateral scan
directions (kernel size: 30 µm for guinea pigs and 150 µm for pigs and
humans) for each of the three input states. The Stokes vectors of each
spectral bin were modelled as μ = DLs, where s is the probing matrix of
the three input states, and the 4 × 3 μ matrix is composed of the three
averaged Stokes vectors. D is a general depolarizing matrix. L is a non-
depolarizing, so-called Jones-derived Mueller or pure Mueller matrix,
representing the retardance and diattenuation to be recovered. Note
that L has only 7 free independent parameters, while μ has 12. Although
insufficient to fully recover D, we corrected its estimated effect on μ by
polarizing the Stokes vectors composing μ to recover L, as described
in detail in Supplementary Method 1.
Combining spectral bins
For each spectral bin, the Mueller matrix L describing the cumulative
round-trip through the sample and the system was recovered as
described above. Next, the polarization reciprocal symmetry of
round-trip optical transmission was recovered for each bin (Supple-
mentary Method 4). The remaining constant alignment of these
matrices to the central spectral bin, described by similarity transforma-
tion with a pure 4 × 4 Mueller matrix, was solved by minimizing the
alignment error of adjacent bins using 10 frames randomly sampled
from a volume scan (Supplementary Method 5). After the alignment,
the reconstructed Mueller matrices from the 9 bins were averaged
elementwise to obtain a general Mueller matrix image
M(x,z)
, where
x
and
z
are the coordinates along the fast scan axis and depth, respec-
tively. Although the initial matrices of the individual spectral bins are
pure Mueller matrices, averaging introduces depolarization, which was
removed using polar decomposition72:
M(x,z)=M△(x,z)MR(x,z)MD(x,z)
,
where
M△(x,z)
is a depolarizer,
MR(x,z)
is a retarder and
MD(x,z)
is a
diattenuator. Combined,
MP(x,z)=MR(x,z)MD(x,z)
defines the pure
Mueller matrix of the cumulative round-trip to sample depth
z
.
System and corneal birefringence compensation
The cumulative round-trip pure Mueller matrix of the retinal surface,
S(x)
, was identified as a function of the lateral position. The matrix
C(x)
,
representing the single-pass linear retardance and diattenuation effect
of transmission through the cornea and the anterior eye to the retinal
surface, was obtained by taking the square root of
S(x)
. It is critical to
unwrap the exponential generator of
S(x)
to force continuity of the
corneal retarder and diattenuator not only in the x direction but also
in the x–y plane. The transmission through the system and the cor-
nea was then compensated by
MPC(x,z)=C−
−
−1
1
1(x)MP(x,z)C−
−
−1
1
1(x)
, where
MPC(x,z)
is the compensated cumulative round-trip Mueller matrix
(Supplementary Method 6). Any single-pass circular retardance and
diattenuation cancels on the round-trip, evading S(x), and remains
uncompensated.
Local birefringence reconstruction
Polar decomposition was further applied to remove diattenuation
M
DC
(x, z) as
MPC (x,z)=MRC (x,z)MDC (x,z)
. The local Mueller matrix
image m(x, z) was reconstructed recursively along depth from
MRC (x,z)
73 as follows:
mxz

m󰁝1xzdzm󰁝1xz
0
M
RC
xzm󰁝1xz
0
m󰁝1xzdz
where
m(x,z0)=√MRC (x,z0)
represents the first row of pixels of the
local Mueller matrix image. The depth-resolved optic axis orientation
and birefringence were then extracted from
m(x,z)
. We used an acry-
lonitrile butadiene styrene phantom to validate the depth-resolved
optic axis reconstruction (Supplementary Method 7).
TRIPS-OCT imaging of pig and guinea pig eyes
The use of animals for these studies was approved by the Institutional
Animal Care and Use Committee of SingHealth (AAALAC Accred-
ited; 2018/SHS/1441, IACUC 1290). All procedures adhered to the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research. In the studies using guinea pig and pig models, the animals
were anaesthetized with an intramuscular injection of a cocktail of
ketamine hydrochloride (27 mg kg−1) and xylazine hydrochloride
(0.6 mg kg−1). The scan was performed with a field of view of 22°,
corresponding to a ~9 × 9 mm area in the pig eye and a 4 × 4 mm
area in the guinea pig eyes. The scanning was performed centred on
the ONH by positioning according to a preview of the OCT B-scans.
The volume scan comprises 1,000 × 1,000 × 3 A-scans over a
square area, with 3 repetitive B-scans on the same location for the
triple-input modulation.
Pig eye TRIPS-OCT imaging and PLM histology
The left eye of a 1-yr-old pig (Yorkshire-Landrace cross, male, National
Large Animal Research Facility, Singapore) was scanned using
TRIPS-OCT. After TRIPS-OCT imaging, the pig was euthanized with
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an overdose of sodium pentobarbital (80–100 mg kg−1) and the left
eye globe was collected. The entire globe was fixed in 10% formalin
for 24 h. After fixation, the eye was transferred to phosphate buffered
saline (PBS). The posterior pole region centred on the ONH was cryo-
sectioned transversely into 30-µm-thick sections and mounted on glass
slides without staining. Fifty sections were obtained and imaged using
a customized polarized light microscope.
Longitudinal guinea pig model
Twenty-one guinea pigs (Elm Hill Labs, female n = 13, Chelmsford),
including albino (n = 17) and pigmented (n = 4) strains, were bred on
site. The animals were reared under a 12 h light/12 h dark cycle with
lights on at 08:00 in the animal-centre facilities. The animals had free
access to standard food and water. Fresh vegetables were provided
twice a day. Refraction data were collected from 1 to 8 weeks using
streak retinoscopy and were reported as the spherical equivalent refrac-
tive error (SE). Retinoscopy was performed on cycloplegic eyes in alert
animals. TRIPS-OCT imaging was performed weekly on both eyes of
the animals.
Adult guinea pig model
Two albino guinea pigs (Elm Hill Labs, female n = 1, Chelmsford) were
selected for TRIPS-OCT imaging from a group of breeders in our animal
facility. The selection criteria for these animals were as follows: (1)
older than 1.5 yr, (2) with clear and healthy eyes without any evidence
of anterior segment or retinal changes and (3) emmetropia (SE = 0D)
or high myopia (SE ≤ −6D). Retinoscopy was performed on cycloplegic
eyes in alert animals. TRIPS-OCT imaging was performed on the eyes
meeting these inclusion criteria.
Guinea pig TEM
Three guinea pigs (Elm Hill Labs, male n = 3, Chelmsford) aged 1,
12 and 16 weeks were killed for TEM histology analysis. The ani-
mals were euthanized with an overdose of sodium pentobarbital
(80–100 mg kg
−1
). After in vivo TRIPS-OCT imaging, the eye globes
were collected and immersed in 0.05 M cacodylic acid sodium and
2.5% glutaraldehyde with PBS (pH 7.4) for 2 h. Then, the cornea and
lens were dissected. A 2 × 2 mm section of scleral tissue from the
superior region of the ONH of each eye was sampled and postfixed
in 1% osmium tetroxide with PBS (pH 7.4) for 1 h at 4 °C, stained with
1% uranyl acetate with double distilled water for 2 h, rinsed and dehy-
drated in graded acetone before embedding in Araldite. Micrographs
of histologic 100-nm-thick cross-sections were imaged using a trans-
mission electron microscope (JEM-2100, Jeol). Electron micrographs
were taken from the sclera region at magnifications of ×100, ×1,000
and ×10,000.
Human recruitment
All procedures performed were in adherence with the ethical stand-
ards of the SingHealth Centralized Institutional Review Board (CIRB
Ref No. 2021/2592). Written informed consent was obtained from
all participants in accordance with the Declaration of Helsinki. The
recruitment was conducted under two cohorts. From the cohort of
normal participants, 80 normal adult volunteers without any ocular
diseases were recruited. The inclusion criteria were as follows: age 21 yr
and above; no diabetes and free from clinically relevant eye disease
that interferes with the aim of the study including glaucoma, diabetic
retinopathy, age-related macular degeneration, uveitis or vascular
occlusive diseases.
From the cohort of participants with pathologic myopia, 10 adult
patients diagnosed with pathologic myopia with staphyloma were
recruited. The inclusion criteria were as follows: age > 21 yr; staphyloma
observed in both eyes on wide-field OCT imaging. The exclusion criteria
consisted of eye conditions that might result in poor-quality imaging
scans (severe cataract, corneal haze/opacity).
Autorefraction, axial length and TRIPS-OCT imaging
In the cohort of normal participants, cycloplegia was achieved using 3
drops of 1% cyclopentolate administered 5 min apart, and cycloplegic
autorefraction was measured 30 min after the last drop using a Canon
RK-F1 autorefractor (Canon). Five readings, all of which were less than
0.25 D apart, were averaged. The SE was calculated as the sphere plus
half cylindrical power. For those who had undergone refractive sur-
gery, data were obtained from records before the surgery. The AL was
obtained using a Zeiss IOL Master (Carl Zeiss Meditec). Five readings,
all within 0.05 mm or less, were averaged. TRIPS-OCT scans were per-
formed on both eyes with 700 × 700 × 3 A-lines in a region of 8 × 8 mm
centred on the ONH.
In the cohort of participants with pathologic myopia, AL was
obtained using Zeiss IOL Master (Carl Zeiss Meditec). Autorefraction
was not performed due to low accuracy in such patients. TRIPS-OCT
scans of both eyes with 700 × 700 × 3 A-lines in a region of 8 × 8 mm
centred on the ONH were performed twice, with the vertical and hori-
zontal directions as the fast and slow scan directions, respectively.
TRIPS-OCT image processing
TRIPS-OCT data acquisition was controlled by an interface software
developed using NI LabVIEW (2020, National Instruments). TRIPS-OCT
images, including those of animal models and humans, were recon-
structed with contrasts of the intensity, birefringence and optic axis.
The cross-sectional intensity images of human eyes were averaged over
12 adjacent B-scans, followed by histogram equalization to enhance
the contrast of the sclera. Cross-sectional birefringence images were
synthesized in the hue, saturation, value (HSV) colour model, with the
H and S channels encoding the birefringence value and the V channel
encoding the intensity value. En face birefringence images were con-
structed as single-channel images cast into the birefringence colour
map with a constant V channel. Cross-sectional and en face optic axis
images were synthesized in the HSV colour model, with the H and S chan-
nels encoding the optic axis orientation and the V channel encoding the
birefringence value. In optic axis images, pixels with intensity SNR lower
than 1 dB were set to background and were replaced with black colour.
Birefringence quantification
Guinea pigs. To obtain en face birefringence images in guinea pigs, the
depolarization index was calculated from the reconstructed general
Mueller matrix M(x, z) and a threshold of 0.9 to define a mask for remov-
ing the background (Supplementary Method 8). A 30 µm vertical-line
kernel was used to filter each cross-sectional birefringence image, fol-
lowed by a maximum projection along the depth. The en face image was
converted to polar coordinates around the ONH. The overall PSB value
was obtained by a maximum birefringence projection along the radial
direction, followed by averaging along the circumferential direction.
Humans. In each cross-sectional image, 20 points were initially manu-
ally placed at the choroidal–scleral interface with a spline interpolation
to define the segmentation. The labeller was free to add more points
and define a finer segmentation by means of an interactive interface
(Supplementary Method 7). Labelling was solely based on the inten-
sity image and performed by a postdoc OCT expert. A 200 µm slab on
the birefringence image, centred on the choroidal–scleral interface
100 µm above and 100 µm below to include fine structures on the
scleral surface, was summed to project onto the en face direction
(Supplementary Method 8). In the projected en face image, the ONH
and fovea were manually labelled. In polar coordinates with the ONH
as the origin, 0° was defined as the vector pointing to the fovea. OPSB
was defined as the mean birefringence value of the annular area centred
on the ONH, with inner and outer circle radii defined as 0.3 and 0.7 of
the ONH–fovea distance, respectively. The annulus was evenly divided
into 12 radial segments. The PPSB was defined as the mean value of the
segment located between the ONH and the fovea.
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Article https://doi.org/10.1038/s41551-023-01062-w
Statistical analysis. As this study of PSB was a pilot study, no sample
size calculation was performed for the animal experiments owing
to the lack of previous studies. Human sample size estimation was
based on the evaluation of the correlation between refractive error
and TRIPS-OCT measurements with 90% statistical power using pre-
liminary parameters from the longitudinal guinea pig study. Analyses
of the correlations between scleral birefringence and other biometrics
were performed by univariate linear regression. Correlation signifi-
cance analyses were performed by conducting an F-test on the linear
model. Significance analyses for scleral birefringence changes were
performed by the Mann–Whitney U test. Inter-eye correlation in the
same participant was addressed by cluster bootstrapping. Specifi-
cally, to determine the CIs and P values of related statistics, a random
resampling process with the original sample size was performed with
replacement at the participant level, and the process was repeated
5,000 times, generating distributions of related statistics. Estimation
of statistics was derived from the median of the generated distribution,
and the 95% CI of the statistics was derived from the 2.5th and 97.5th
percentiles. All analyses were performed using MATLAB (R2019b,
R2020b, R2021b, MathWorks).
Reporting summary
Further information on research design is available in the Nature Port-
folio Reporting Summary linked to this article.
Data availability
Processed animal data (shown in Figs. 3 and 4), including en face images
and refractive errors, are available from figshare
74
. Additionally, one
example guinea pig B-scan modulated by triple polarization states
(shown in Fig. 1) is also available from figshare
74
. The entire raw dataset
of animal experiments is more than 25 TB in size and can be shared
upon request with appropriate data transfer methods. For the clinical
study, the raw data acquired during the study are available for at least 5
years from the corresponding author on reasonable request, subject to
approval from the SingHealth Centralised Institutional Review Board.
A request will be processed within 3 months.
Code availability
The central algorithm to reconstruct TRIPS-OCT images from
triple-measured Stokes vectors can be found at https://github.com/
DrXinyu/TRIPS-OCT.
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Acknowledgements
We thank J. M. Busoy and K. J. V. Catbagan of the Singapore Eye
Research Institute for keeping and handling the animals; Q. Hu,
B. Kulantayan, H. Chye, J. L. H. Tay and S. B. J. Pow of the Singapore
Eye Research Institute for coordinating with patients; J. Gnalian
of the University of Pittsburgh for performing the PLM histology;
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Article https://doi.org/10.1038/s41551-023-01062-w
L. Liu of Nanyang Technological University, J. Ren of Harvard
Medical School and T. Ling of Nanyang Technological University for
discussion on data presentation; and C. Zhang of Tsinghua University
for discussion on statistics. This work was funded by grants from the
Industry Alignment Fund - Industry Collaboration Projects (IAF-ICP)
Grant (I1901E0038, L.S., Q.V.H., A.W.C., R.P.N., V.A.B., M.A. and
S.-M.S.) and Johnson & Johnson Vision. We also acknowledge the
support of the National Medical Research Council (CG/C010A/2017_
SERI, L.S.; OFLCG/004c/2018-00, L.S.; MOH-000249-00, J. Chua;
MOH-000647-00, L.S.; MOH-001001-00, L.S.; MOH-001015-00,
L.S.; MOH-000500-00, L.S.; MOH-000707-00, L.S.; MOH-001072-
06, L.S.; NMRC/CSIRG/MOH-000531/2021, Q.V.H.); the National
Research Foundation Singapore (NRF2019-THE002-0006, L.S. and
NRF-CRP24-2020-0001, L.S.), A*STAR (A20H4b0141, L.S., J. Chua),
the Singapore Eye Research Institute & Nanyang Technological
University (SERI-NTU Advanced Ocular Engineering (STANCE)
Program, L.S.), the SERI-Lee Foundation (LF1019-1, J. Chua), the US
National Institutes of Health (P41EB-015903, M.V. and R01 EY023966,
I.A.S.), the EU (H2020-MSCA-IF-2019 program 894325, M.L.) and the
Singapore Eye Research Institute & National University of Singapore
ASPIRE Program (NUHSRO/2022/038/Startup/08, R.P.N.).
Author contributions
X.L. developed the TRIPS-OCT system. L.J., Q.V.H., X.L., M.K., R.P.N.,
M.L., L.S. and V.A.B. designed and conducted the animal studies.
I.A.S. and X.L. conducted the PLM analysis. X.L., M.K., J. Chua, Q.V.H.,
A.W.C., B.T., J. Cheong, V.B., R.S.C., M.J.A.G., M.A., S.-M.S. and L.S.
designed and conducted the human studies. M.V. and X.L. developed
the TRIPS-OCT reconstruction method. L.S., X.L., M.V., R.P.N., G.G. and
I.A.S. interpreted the data. L.S. conceived the overall idea, obtained
funding and supervised the entire study. All authors read and edited
the manuscript.
Competing interests
X.L. and L.S. are inventors on a provisional patent (10202009128V,
Singapore, 2020) related to TRIPS-OCT technology. The other authors
declare no competing interests.
Additional information
Extended data is available for this paper at
https://doi.org/10.1038/s41551-023-01062-w.
Supplementary information The online version
contains supplementary material available at
https://doi.org/10.1038/s41551-023-01062-w.
Correspondence and requests for materials should be addressed to
Leopold Schmetterer.
Peer review information Nature Biomedical Engineering thanks
Johannes de Boer, Shaohua Pi, Ruikang Wang and the other,
anonymous, reviewer(s) for their contribution to the peer review of this
work. Peer reviewer reports are available.
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Extended Data Fig. 1 | Triple-input PS (TRIPS) OCT. a, TRIPS-OCT system
schematic. Briefly, compared to a conventional OCT system that records only the
intensity of the light, the polarization states of light are additionally recorded
by a polarization diversity detection circuit (BS, PBSs, D1, D2). The triple-state
modulator was inserted into the sample arm. b, Triple-state modulator. The
modulator comprises a polarizer and an EOM. c, EOM modulation scheme.
The EOM is driven by a triple-step voltage to produce three retardance values,
−120°, 0°, and 120°. d, Polarizer axis configuration. The angle between the
linear polarizer preceding the EOM and the optic axis of the EOM is set as 27.37°.
e, Mutually orthogonal polarization states on the Poincaré sphere resulting from
triple-state modulation. FC, Fibre coupler. P1-2, Polarizer. A, Motorized aperture.
C1-2, Circulator. PBS, Polarizing beam splitter. BS, Nonpolarizing beam splitter.
SG, Scanning galvo mirrors. EOM, Electro-optic modulator. M, Mirror. D1-2,
Photodetector.
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Extended Data Fig. 2 | Comparison between dual-input and triple-input PS-
OCT (TRIPS-OCT) methods under the condition of the same acquisition time.
a, Nine repetitive scans modulated by triple polarization states of the guinea
pig retina were acquired in-vivo. Different sets of 6 of the 9 repetitive scans were
used to reconstruct the birefringence images with the dual-input method and
TRIPS-OCT, respectively. For the dual-input method, the three scans modulated
by the same input polarization state were averaged before birefringence
reconstruction. For the triple input method, the two scans modulated by the
same polarization state were averaged before birefringence reconstruction. The
averaging was performed on the intensity images without considering the phase.
This averaging process confirms that the acquisition time of the data used by
the dual-input and triple-input methods for the birefringence reconstruction
are identical. b, Birefringence images reconstructed from triple-input and
dual-input methods using different combinations of the input polarization
states. c, Birefringence noise characterization for the different combinations
using the inner retina in Fig. a indicated by the orange area (pixel number n = 5117
from 1 cross-sectional image). Central lines of violin plots indicate mean. The
improvement in noise performance using TRIPS-OCT is quite consistent between
different combinations of Stokes vectors. The slight difference in the noise level
of dual-input combinations is due to the dependency between the edge-artifacts
and the absolute polarization states (Supplementary Discussion 1). SD: Standard
deviation.
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Extended Data Fig. 3 | Noise analysis for PS-OCT. a, Simulation comparing
TRIPS and dual-input methods on a four-layer sample. Black arrows indicate
edge-artifacts in the dual-input reconstruction method, absent in TRIPS-OCT.
The artifacts are pronounced at brick-wall-jumps in the scattering profile. b,
TRIPS vs. dual-input reconstruction of an A-scan of a guinea pig retina from the
B-scan in Fig. 1a. Black arrows indicate the observed edge-artifacts. c, Numerical
model studying edge-artifacts and intensity profile variations. The sample
is modelled by two nonbirefringent scattering layers under a transparent
birefringent layer with random optic axis and a retardance of 20°. Intensity
variation is created by changing the reflectivity of the scatterers within each
layer. d, Numerical model studying birefringence noise and intensity signal-
to-noise ratio (SNR). The sample is modelled by a nonbirefringent scattering
layer. White noise is added to the simulated fringes to create different SNRs. e,
Numerical model studying birefringence noise and axial motion. Axial motion
is modelled by a random shift of the sample along depth with a zero mean and
a standard deviation σ. Scattering layers are modelled by scatterers embedded
within nonbirefringent or birefringent media, creating fully developed speckle
patterns in OCT scans. OCT scans are simulated by generating the fringes in
wavenumber domain of the individual scatterers and then transforming the
summed fringes into the depth domain using Fourier transformation with a
resolution of 6 micrometres. Sixteen scans with independent speckle patterns
are averaged to suppress the speckle noise before proceeding to birefringence
reconstruction. Birefringence reconstruction (Supplementary Method 2)
is performed by TRIPS, dual-input geometric reasoning, Jones matrix and
single-input geometric reasoning. Data are presented as mean values +/− 95%
confidence intervals, which are created by bootstrapping with n = 500 repetitive
simulations on random optic axis and random positioning of the scatterers.
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Extended Data Fig. 4 | Repeatability of scleral birefringence measurements
in guinea pigs using TRIPS-OCT. a, Twelve guinea pig eyes were imaged
repetitively with identical imaging angles. b, Linear correlation of mean values
of repetitively measured birefringence. c, Bland–Altman plot of the repetitively
measured birefringence. d, Repeatability test under varying imaging angles. To
test the repeatability under slightly varying imaging angles, TRIPS-OCT imaging
was performed repetitively on guinea pigs placed in both the prone and supine
positions. Cross-sectional images indicate slightly varying retina tilt. Blood
vessels indicated by white arrows are used to register the rotated birefringence
images. e, Twelve eyes were imaged repetitively with prone and supine
positions. f, Linear correlation of the mean values of the repetitively measured
birefringence. g, Bland–Altman plot of the repetitively measured birefringence.
The r values are calculated by Pearson correlation. The p values are calculated
by F-test against a constant model. Excellent repeatability was achieved under
slightly varying imaging angles because the retardance and diattenuation of the
cornea were correctly compensated for in the reconstruction. The 1.96 SD error
was 6.1% under different imaging angles, higher than the value of 2.4% under
identical imaging angles, perhaps due to the slight change in scleral thickness
under different tilts. SD: Standard deviation.
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Extended Data Fig. 5 | Data summary and analysis in the human study. Flow
diagram summarizing the human subjects and eyes included and excluded in
the analysis. Eyes included in the analysis were grouped into three groups (more
characteristics are provided in Supplementary Table 1). Group 1: Emmetropia
or low myopia (3 D ≤ SE< −6 D) with no pathological conditions, 69 eyes from
42 subjects (23 female) with a mean age of 41.29 years, a mean SE of −1.74 D, and
a mean AL of 24.44 mm. Group 2 : High myopia (≤ −6D) with no pathological
conditions, 16 eyes from 9 subjects (8 female) with a mean age of 39.20 years, a
mean SE of −7.72 D, and a mean AL of 26.88 mm. Group 3 : Pathologic myopia with
staphyloma, 15 eyes from 9 subjects (6 female) with a mean age of 58.22 years and
a mean AL of 29.05 mm. SE, Spherical equivalent refractive error. AL, Axial length.
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Extended Data Fig. 6 | Scleral collagen fibre orientation and birefringence in
adult guinea pigs without and with myopia, as assessed with TRIPS-OCT. a,
Images of two adult guinea pig eyes without and with myopia. En face intensity
images are obtained from an average projection along the depth. En face optic
axis images are obtained from the outer layer of the sclera after flattening the
images using the surface of the retina. White dotted lines indicate the locations
of the cross-sectional images. The sclera is manually segmented in the cross-
sectional images, and two regions of interest (ROIs) are selected in each image
for localized comparison. The locations of cross-sectional image are roughly
matched by the same relative position according to the optical nerve head.
From the cross-sectional images, we observe thinning of the sclera tissue and
deformation of the eye shape towards axial elongation in the highly myopic eye
(−9 D). b, Histograms of measured birefringence and collagen fibre orientation in
the entire sclera (left panel, pixel numbers n = 10399, 8477 for each region), ROI 1
(middle panel, pixel numbers n = 1269, 898 for each region) and ROI 2 (right panel,
pixel numbers n = 1330, 784 for each region) from the cross-sectional images.
Comparing the birefringence measurements, the average scleral birefringence
increases in the myopic eye. The interweavement of collagen fibre, however,
decreases as evidenced by that the local maxima in the angular histograms of
the myopic eye are higher as compared to the emmetropic eye. As a result of
scleral collagen remodelling, the increase in PSB in adult guinea pigs with myopia
may be due to the augmented collagen fibre alignment and the reduction of
interwoven fibres. Scale bars, a, vertical: 300 µm, horizontal: 1 mm.
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Extended Data Fig. 7 | Scleral collagen fibre orientation and birefringence
in a young guinea pig at ages of 2 and 8 weeks, as assessed with TRIPS-OCT.
a, Images of a guinea pig eye at ages of 2 weeks and 8 weeks, respectively. En
face intensity images are obtained from an average projection along the depth.
En face optic axis images are obtained from the inner layer of the sclera after
flattening the images using the surface of the retina. White dotted lines indicate
the locations of the cross-sectional images. The sclera is manually segmented in
the cross-sectional images, and two regions of interest (ROIs) are selected in each
image for localized comparison. ROIs are registered by choroidal vessel patterns,
indicated in a by yellow arrows. b, Histograms of measured birefringence and
collagen fibre orientation in the entire sclera (left panel, pixel numbers n = 10717,
15096 for each region), region of interest 1 (ROI 1, middle panel, pixel numbers n
= 1981, 2615 for each region) and ROI 2 (right panel, pixel numbers n = 2772, 3732
for each region) from the cross-sectional images. Comparing the measurements
at ages of 2 and 8 weeks, the average scleral birefringence increases with aging,
whereas the distribution of collagen fibre orientation broadens as evidenced by a
reduction of the local maxima and an increase of the local minima in the angular
histograms. Broadening of the fibre orientation distribution may be due to an
increase in fibre interweavement, the increase in the interwoven fibre diameters,
or a combined effect of both. Scale bars, a, vertical: 300 µm, horizontal: 1 mm.
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Extended Data Fig. 8 | Scleral collagen fibre orientation and birefringence of
the macular region in humans, as assessed with TRIPS-OCT. a, Cross-sectional
scans of the macular region of human subjects with different degrees of myopia.
b, Histograms of measured birefringence and collagen fibre orientation within
the regions of interest (ROIs, pixel numbers n = 2032, 2554, 2079 for each region)
from the cross-sectional images. The ROIs are selected as an area in the sclera
below the fovea measuring 100 µm vertically and 2 mm laterally, extending from
the choroidal-scleral interface. The boundaries of the ROIs are indicated by the
dotted lines in the images. The choroidal-scleral interface is labelled manually.
We observe that the average scleral birefringence is higher in patients with a
higher degree of myopia, as well as an increase in the local maxima and a decrease
in the local minima in the angular histograms of collagen fibre orientation.
Specifically, in the eye with moderate myopia, there is a reduction in fibres
oriented at 150° compared to the emmetropic eye. In the eye with high myopia,
the collagen fibres at 60° completely disappear in the ROI. From this observation,
we suppose that the increased PSB in patients with myopia is due to a decrease of
the interweavement of the collagen fibres. Histogram equalization is applied to a
(upper panels). Scale bars, a, vertical: 300 µm, horizontal: 1 mm.
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Extended Data Table 1 | Univariate linear regression analysis of posterior pole scleral birefringence with multiple variables
within the eyes in the emmetropia or low myopia (3 D ≤ SE< –6 D) group
Linear regression models are itted using data from 69 eyes from 42 individuals. 95% conidence intervals of B and r are calculated by cluster bootstrapping. The r values are calculated by
Pearson correlation. The p values are calculated by F-test against a constant model. B, Model gradient, r, Pearson’s coeficient, CI, Conidence interval.
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Last updated by author(s): Mar 23, 2023
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Software and code
Policy information about availability of computer code
Data collection NI LabVIEW 2020 was used to develope the data acquisition and control software.
Data analysis We used MATLAB (R2019a, R2020b, R2021b) to reconstruct the TRIPS-OCT images from the photodetector readout. We used MATLAB
R2021b to segment the TRIPS-OCT images, quantify the measurements, and conduct the statistical analysis. The central algorithm to
reconstruct TRIPS-OCT images from triple measured Stokes vectors can be found at https://github.com/DrXinyu/TRIPS-OCT.
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Processed animal data (shown in Figs. 3 and 4), including en-face images and refractive errors, are available from figshare at https://doi.org/10.6084/
m9.figshare.21300576. Additionally, one example guinea pig B-scan modulated by triple polarization states (shown in Fig. 1) is also available from figshare. The
entire raw dataset of animal experiments is more than 25 TB in size and can be shared on request with appropriate data-transfer methods. For the clinical study, the
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size As this study of PSB was a pilot study, no sample-size calculation was performed for the animal experiments, owing to the lack of previous
studies. Empirically we decided to use 21 guinea pigs to investigate the correlation between refractive error and scleral birefringence. In the
clinical study (Figs. 5 and 6), the sample-size calculation was based on the evaluation of the correlation between refractive error and TRIPS-
OCT measurements with a 90% statistical power using preliminary parameters from the longitudinal guinea-pig study. For other experiments
aiming for validation of the technology of TRIPS-OCT, biological independence is not required. Therefore, one animal was used for each
specific aim.
Data exclusions In the longitudinal guinea-pig study (Figs. 3 and 4), no data were excluded. In the clinical study (Figs. 5 and 6), we excluded 75 eyes (47%) with
suboptimal positioning (50 eyes, 31%) and insufficient signal-to-noise ratio (average scleral SNR < 4.6 dB) from the sclera (25 eyes, 16%). The
exclusion of images with insufficient intensity signal was pre-established. The threshold 4.6 dB was determined after the study because no
preliminary TRIPS-OCT data were available. Perfect positioning of the scan was not always guaranteed in the clinical study because the
positioning of TRIPS-OCT imaging head was not always accurate, owing to the lack of pupil alignment camera, fundus camera, eye tracker, or
automatic depth-positioning unit. A sufficient signal from the sclera was not always guaranteed in the clinical study because a thick choroid
may limit the light penetration to the sclera.
Replication For the TRIPS-OCT technology, at least 294 volume scans (42 eyes, 7 weeks) were performed in the guinea-pig eyes in the longitudinal animal-
model study; 180 volume scans were performed in human eyes, from which 100 volume scans were rated as high-quality, defined by the
quality criteria in the human study. For the analysis of the correlation between scleral birefringence and the degree of myopia, no replication
was performed because all validated data are included to achieve maximum statistical power.
Randomization No randomization was applied because the study was focused on the correlation between TRIPS-OCT measurements and the degree of
myopia.
Blinding For the animal study, blinding was not possible because there was no prior knowledge of the association or correlation between refractive
error and scleral birefringence. In the human study, The investigators were blinded to the birefringence measurements when segmenting the
images, and were blinded to myopia status of the subjects during the processing of the TRIPS-OCT measurements.
Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Materials & experimental systems
n/a Involved in the study
Antibodies
Eukaryotic cell lines
Palaeontology and archaeology
Animals and other organisms
Human research participants
Clinical data
Dual use research of concern
Methods
n/a Involved in the study
ChIP-seq
Flow cytometry
MRI-based neuroimaging
Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals A 1-year-old pig (Yorkshire-Landrace cross, Male, National Large Animal Research Facility, Singapore) was euthanized for TRIPS-OCT
and PLM imaging. Three guinea pigs (Elm Hill Labs, Male n = 3, Pigmented n = 1, Chelmsford, US) aged 1, 12, and 16 weeks were
euthanized for TEM histology analysis. 21 guinea pigs (Elm Hill Labs, Albino n = 17, Female n = 13, Chelmsford, US) were bred on-site
for refraction-development analysis. 2 guinea pigs (Elm Hill Labs, Albino n = 2, Female n = 1, Chelmsford, US) aged 1.6 and 2.1 years
were selected for TRIPS-OCT imaging from a group of breeders in our animal facility.
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3
nature portfolio | reporting summary March 2021
Wild animals The study did not involve wild animals.
Field-collected samples The study did not involve samples collected from the field.
Ethics oversight The use of animals for these studies was approved by the Institutional Animal Care and Use Committee of SingHealth (AAALAC
Accredited; 2018/SHS/1441, IACUC 1290).
Note that full information on the approval of the study protocol must also be provided in the manuscript.
Human research participants
Policy information about studies involving human research participants
Population characteristics The population characteristics of each eye group used in the analysis are described in Supplementary Table 1.
Recruitment 80 normal adults aged 21 years and above without any known ocular diseases were recruited. The inclusion criteria were as
follows: age 21 years and above; no diabetes and free from clinically relevant eye disease that interferes with the aim of the
study, including glaucoma, diabetic retinopathy, age-related macular degeneration, uveitis, or vascular occlusive diseases.
10 patients diagnosed with pathologic myopia with staphyloma were recruited. For patients with pathologic myopia, the
inclusion criteria were as follows: age > 21 years; both eyes presented staphyloma under wide-field OCT imaging. The
exclusion criteria were as follows: eye conditions that may potentially result in poor quality imaging scans (severe cataract,
corneal haze/opacity).
Normal adults were recruited from patients, visitors and staff of the Singapore National Eye Center. Patients with pathologic
myopia were referred by clinicians of the Singapore National Eye Center. As a pilot study conducted in Singapore, 91% of the
participants were Asian. Participants were compensated with 30 Singapore dollars each.
Ethics oversight All procedures performed were in adherence with the ethical standards of the SingHealth Centralized Institutional Review
Board (CIRB Ref No. 2021/2592). Written informed consent was obtained from all participants in accordance with the
Declaration of Helsinki.
Note that full information on the approval of the study protocol must also be provided in the manuscript.
Clinical data
Policy information about clinical studies
All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.
Clinical trial registration CIRB Ref No. 2021/2592. There is no public registration because the clinical study was not interventional.
Study protocol Polarization Sensitive Optical Coherence Tomography — Phase II: A Pilot Study (R1819/61/2021).
Data collection The data of patients and healthy individuals were collected between 15/02/2021 and 28/05/2021 from the research clinic in the
Singapore Eye Research Institute. Healthy individuals were recruited from patients, hospital staff and visitors. Patients with
pathologic myopia were recruited from general clinics in the Singapore National Eye Center and refereed by clinicians. Auto-
refraction, axial length, age, sex and TRIPS-OCT data were extracted from the data-management unit of the institute.
Outcomes Outcomes are not relevant because the study was not interventional.
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