A Study to Test the Diagnostic Potential of Brillouin Microscopy for Corneal Ectasia
Conditions: Keratoconus, Ectasia, Crosslinking, Fuchs' Endothelial Dystrophy
Sponsor: Massachusetts General Hospital

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Identifiers: NCT02118922   Unique Protocol ID: 2012P002178 
Brief Title: A Study to Test the Diagnostic Potential of Brillouin 
Microscopy for Corneal Ectasia 
 
I. BACKGROUND AND SIGNIFICANCE  
A) HISTORICAL BACKGROUND 
The overall goal of this study is to change the current paradigm to detect corneal ectasia. We aim to 
establish biomechanical criteria to identify early signs of keratoconus and to screen at-risk subjects 
for post-LASIK ectasia based on a novel Brillouin optical imaging. 
 
Ectasia refers to thinning and bulging of the cornea, which causes 
severe visual degradation and frequently requires a corneal 
transplantation [1]. The mechanism of ectasia is known to relate to 
corneal biomechanics (Fig. 1). In a healthy cornea, collagen fibers 
provide the mechanical strength to withstand the outward intraocular 
pressure (IOP); if the corneal tissue becomes compromised and 
weakened, the ectasia can develop. 
 
An abnormal loss of corneal integrity and strength can either result 
from degenerative ocular conditions, such as keratoconus, or as a 
complication of refractive surgery, most notably with Laser In Situ 
Keratomileusis (LASIK).  
Keratoconus is the most common corneal degeneration in the US 
affecting ~1/2000 of the general population [2] with a mean onset age 
of 15.4 years [3]. LASIK is the most frequently performed laser surgery in the US with nearly 1 
million operations per year. Iatrogenic ectasia following LASIK is the most feared complication of 
surgery causing severe visual degradation and often requiring a corneal transplantation because of 
the irreparable corneal changes after the vision correction [1].  
For these conditions, the early diagnosis is crucial. Keratoconus progression could be halted by 
promising interventions, such as corneal collagen crosslinking [4], if detected early. In addition, 
patients with weak corneas could be screened and counseled to avoid LASIK surgery. Ectasia due 
to keratoconus or after LASIK have similar biomechanical presentations [5]. In fact, unidentified 
keratoconus is the most likely cause for post-LASIK ectasia occurrence [6], and the early-diagnosis 
of keratoconus onset is widely considered as the key advance needed to dramatically reduce the 
occurrence of post-LASIK ectasia [6]. 
Current diagnostic methods are based on morphological rather than biomechanical analysis. 
Current diagnosis of keratoconus is clinical. The irregular morphologic patterns of the cornea can 
be detected by pachymetry and topography before clinical signs occur, but these tests cannot reliably 
differentiate truly weak or keratoconic corneas from atypical normal ones [7, 8].  The Screening 
protocols for LASIK that rely on indirect morphological risk factors have had limited success in the 
clinic. About 15% of patients seeking laser vision correction are considered at-risk and are thus 
denied treatment; on the other hand, patients with normal appearing pachymetry and topography 
 
Fig. 1: Intraocular pressure (IOP) 
induces corneal tensile (T) load.  A 
weak cornea, with reduced elastic 
modulus, cannot withstand IOP and 
thus thins and bulges, causing 
ectasia. 

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and no other risk factors have gone on to develop post-LASIK ectasia [9-11]. This situation has also 
led to multimillion dollar lawsuits [12] and has contributed to the decision by the FDA to reevaluate 
LASIK safety [13, 14]. 
The compelling need for improved diagnostics has triggered interest in the mechanical properties of 
the cornea, e.g. elastic modulus or stiffness, as their connection to the pathophysiology of the 
condition makes them highly suited to early identify patients at risk for developing ectasia [6, 15].  
However, mechanical measurements are traditionally performed by macroscopic and destructive 
methods [16]. Recently, a widespread effort has been put forward to achieve non-invasive 
assessment of corneal mechanical properties. Corneal hysteresis measured by an ocular response 
analyzer [17] (ORA) has been shown to correlate with advanced keratoconus [18]. However, 
hysteresis is indirectly related to the elastic properties, and the clinical usefulness of the ORA device 
in early keratoconus patients remains highly questionable [19-21]. Other techniques based on 
ultrasounds [22, 23] are under development [24-27]. However, currently there is no clinical 
ophthalmic device capable of measuring corneal stiffness or elastic modulus.  
Our Innovation: Brillouin microscopy - In this study, we will introduce an entirely novel approach 
for in vivo biomechanical analysis of the cornea via optical means. In the past years, we have 
invented and developed an optical technology, Brillouin microscopy, highly suited for elasticity-
based screening in refractive surgery  [28].  When laser light hits a sample (e.g. lens or cornea), a 
portion of the light is reflected due to the acoustic waves that are weak but naturally present in the 
material by thermal fluctuations. The acoustic waves propagate with a speed that is directly related 
to the sample’s elastic property. We measure the acoustic speed (therefore the elastic modulus) by 
measuring the frequency shift of the reflected light using a specially designed spectrometer, i. e. the 
Doppler principles used in a laser-based speed measuring of a traveling car. The spectrometer we 
have developed can detect the small Brillouin frequency shift 1000 times more efficiently than 
existing instruments. This technique is different from ultrasound and offers much higher resolution 
and sensitivity. It is capable of probing the elasticity of the tissues non-invasively with microscopic 
resolution. We have extensively validated the Brillouin microscopy for the ophthalmic research by 
mapping ocular tissue elasticity, its spatial heterogeneity and relevant changes in both lens [29] and 
cornea [30].  
 
Importantly, using human corneas ex vivo, we have recently demonstrated that Brillouin technology 
can clearly differentiate ectatic from normal corneas based on different elastic modulus. We also 
have developed a prototype instrument for clinical use. We have already tested the safety of the 
prototype system in vivo on five human subjects [31]. This is part of an ongoing safety study 
approved by PHC IRB.  
 
B) PRE-CLINICAL AND CLINICAL STUDIES 
The traditional Brillouin spectroscopy,  widely 
used for material characterization [32], is slow 
in acquiring spectra (10 min to 1 hour per 
spectrum) 
thus 
limited 
to 
point-sample 
analysis. In 2008, we have introduced a novel 
spectrometer, that reduced the acquisition time 
to 1-10 seconds per spectrum [33]. Then with 
the multi-stage VIPA spectrometry [34, 35], we Fig. 2: (a) Cross-sectional Brillouin image of bovine cornea ex
vivo. Color represents the measured Brillouin frequency shift.

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further reduced the spectrum acquisition time by 100 times, which enabled us introducing the 
Brillouin confocal microscopy to obtain the first 3D elasticity imaging of the cornea (Fig. 2) and 
the lens in animals [29, 30].  
As preliminary investigations for this study  
1) We have performed theoretical calculations and modeling on the safety of Brillouin technology. 
Besides we have experimentally tested the validity of our theory and modeling.  
2) In ex-vivo animal studies, we have found that the Brillouin technology reveals depth-dependent 
variations in corneal modulus corresponding to different stromal collagen organizations [30].  
3) Through in vivo longitudinal study in mice, we have tested the safety and efficacy of our 
technology [29].  
4) With ex-vivo human tissue studies we have shown that the Brillouin technology can measure the 
abnormal weakening of human cornea due to ectasia. 
5) We have completed a safety study on 10 heathly human subjects in vivo [31].  
6) In the in vivo human study, we have shown that our current Brillouin instrument provides 
sufficient sensitivity and reproducibility to measure corneal ectasia in humans in vivo. 
1. Safety and risk analysis of the Brillouin technology for use in human.  
Theory - The Brillouin technology uses illumination intensity levels comparable or smaller than 
equivalent ocular instruments that are already FDA-cleared and widely used in the clinical practice 
such as the corneal confocal scanning microscopy and the anterior-segment optical coherence 
tomography. The optical source within the experimental Brillouin instrument emits near-infrared 
light at the wavelength of 780 nm. Exposure of the human eyes to this light will be within safety 
limits established by the American National Standard for Safe Use of Lasers (ANSI Z136.1-2007). 
The ANSI standard indicates that exposure to this light at the levels we propose for imaging (<3 
mW) does not pose a risk to the subjects even for continuous prolonged exposure of 8h. We expect 
to limit the continuous exposure time for one imaging scan to less than 10 seconds, i.e. very short 
within this time limit. The subject will be asked to comply with the imaging procedure 40-50 times 
during the session to gather spatially-resolved data within the cornea. A short break between the 
imaging scans will be provided to rest the eye. The imaging will not require any direct contact with 
the eye. The scanning procedure is not associated with any discomfort, except for the possibility of 
minimal discomfort arising from staring at the low intensity light in order to reduce the eye 
movements to a minimum, and from straining due to not blinking during the scanning. The only 
direct contact will be at the subject's chin and forehead with 
the respective rests of the standard slit-lamp interface. The 
chin and forehead rests will be cleaned with alcohol wipes in 
between the subjects.  
To evaluate the potential risks associated with the use of 
Brillouin ocular microscopy, we can calculate the Maximum 
Permissible Exposure (MPE), defined as the highest power 
or energy density that can be admitted to the eye without 
causing a biohazard. MPE corresponds to 10% of the dose 
that has a 50% chance of creating damage in a worst-case 
scenario. In the Brillouin scanner, the laser light is focused 
in the cornea, and it is well diffused when it reaches the 
retina. According to the International Commission on Non-
Ionizing Radiation Protection (ICNIRP), the exposure limit 
 
Fig. 3: Maximum permissible power for cw 30 
s exposure with NA=0.1.  

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for the cornea-lens thermal safety is 4 W/cm2 for continuous wave sources in the wavelength region 
of 400 - 1050 nm; i.e. MPE = 32 mW in a 1-mm-diameter zone (0.79 mm2 in area) [36]. The 1-mm 
aperture for irradiance averaging is based on the thermal modeling [37], which shows that the 
temperature rise is independent of the beam size up to 1 mm due to rapid thermal conduction 
(dissipation ~ 0.5 W/m/oC) in the cornea and lens. As for the retina, the dominant mechanism of 
retinal damage is thermal for an exposure time (T) longer than 0.25 s. According to the guidelines 
from American National Standard Institute, the exposure limit for the retinal thermal hazard has 
been expressed as 1.8 x 10-3 CA CE T-0.25 [W/cm2], where CA = 1 - 1.5 (for  = 400 - 800 nm), CE = 
267 (for  = 0.2; i.e. NA = 0.1), and the aperture size of 7 mm (area of 0.38 cm2) [38]. The MPE is 
calculated to be 183 mW for T = 1 s and 66 mW for T = 60 s, which is consistent with the above 
analysis. Taken together, we conclude that the Brillouin scan in the cornea and lens does not pose a 
risk to the human eye. 
Experiment - To experimentally test the theoretical prediction, since the lower exposure limit is 
predicted for corneal thermal safety, we used an infra-red high-resolution camera (FLIR SC8000) 
with a temperature sensitivity of 0.1C to 
physically detect any possible changes in 
temperature of the cornea due to light 
illumination. In this setup, we used a 
porcine eyeball and we focused laser light 
of different power levels onto the cornea 
with several objective lenses providing 
numerical apertures (NA) from 0.05 to 0.3. 
At the levels of light exposure we intend to 
use in our pilot study (i.e. ~ 0.5mW, NA = 
0.1), we have detected no increase in 
temperature on the corneal surface, even 
after prolonged exposures of greater than 60 seconds. In order to observe a detectable change in 
temperature, we had to shine 180 mW of light power onto the specimens for 10 seconds, which has 
resulted in a temperature rise less than 1C, well below tissue damage threshold (Fig. 4). These 
results are consistent with thermal modeling prediction. Note that this exposure level is some 300 
times higher than the power level we plan to use in our study. Therefore, we have experimentally 
verified that the thermal modeling predictions are accurate and that the Brillouin technology poses 
no risk to the cornea. 
2. Ex vivo animal studies : 
Brillouin can detect the local depth-dependent elasticity changes due to the corneal 
collagen organization (bovine ex vivo).  
The collagen content and the fibril organization are 
known to be responsible for the mechanical strength 
of the corneal stroma [39-41]. We have investigated 
the correlation between the internal micro-structure 
and the elastic modulus of cornea comparing high-
resolution axial profiles of intact bovine corneas 
(Fig. 5a) with the corresponding collagen-stained 
(Masson’s trichrome) histology images (Fig. 5b) and 
second harmonic generation (SHG) microscopy to 
highlight collagen fibers (Fig. 5c).  The anterior part 
Fig. 4: Corneal temperature maps before (a) and after (b) light exposure 
at 180 mW for 10s, which corresponds to more than 100 times higher 
than the exposure levels proposed in our study. The maximum 
temperature elevation is less than 1C. 
 
Fig. 5:  (a) Brillouin depth profile of an intact bovine 
cornea. (b) Masson’s trichrome stained image of a thin 
tissue slice. (c) A second-harmonic-generation image of 
the section. Scale bar, 0.2 mm.  

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of the stroma shows a markedly interwoven organization of collagen fibers and has the highest 
Brillouin modulus.  
In the posterior and innermost region of the cornea, collagen fibers mostly run parallel to the corneal 
surface and the collagen amount decreases towards the endothelium, which correlates with the 
Brillouin modulus decrease [42]. These results are also consistent with previous mechanical tests 
on shaved-off corneas that showed decreasing elastic modulus across the corneal depth [43]. This 
correlation strongly supports the hypothesis that LASIK surgery, by depleting the cornea of some 
of its stroma with high modulus, will lead to decreased overall modulus and stiffness. The Brillouin 
technology will be sensitive enough to detect such change. 
 
3. In vivo animal studies: 
Safety and efficacy of the Brillouin technique in mice in longitudinal study in vivo.  
A total of 12 mice (C57BL/6 strain) were tested; 
11 mice up to 40 weeks of age were imaged at 
various ages only once each, and a single mouse 
was imaged weekly from day 17 after birth till 9 
weeks later [44]. Figure 6 depicts measured 
Brillouin frequency shift in mouse ocular lens 
tissue, which clearly shows an increasing trend 
with age. We have not observed any complications 
due to imaging. In addition, it is evident that the 
repeated measurements on the same mouse follow 
the trend of the curve obtained on separate mice; 
this further confirms the non-perturbative, non-
disturbing character of our measurements.  
4. Ex vivo human studies : 
Brillouin technique detects lower elastic modulus in ectatic corneas.  
We have tested 4 ectatic (weakened) corneas from keratoconus patients whom underwent corneal 
transplantation, and 5 normal donor corneas. As shown in Fig. 7, the profiles of ectatic vs. normal 
corneas are dramatically different. Compared to normal controls, ectatic corneas have lower mean 
elastic modulus (Fig. 7c) and increased slope, i.e. the rate of elastic modulus drops across depth 
(Fig. 7b). The negative Brillouin slope of ectatic corneas was 0.94 ± 0.15 GHz/mm, showing a 
statistically significant increase from 0.4 ± 0.25 GHz/mm for the controls (unpaired t-test P < 
0.0025); the mean Brillouin modulus was 2.58 ± 0.096 GPa for ectatic corneas and 2.72 ± 0.06 GPa 
for normal corneas (unpaired t-test P < 0.05). Also the stiffness, i.e. the integral under the elasticity 
 
Figure 6: Peak frequency shifts of murine lens nucleus in vivo 
measured from individual mice at various ages (solid circles) 
and one mouse over time (open circles) 
 
Fig. 7: (a) Brillouin profile of human corneas ex vivo obtained from normal and keratoconus/ectasia patients. The differences in 
the slope (b) and the mean modulus (c) are statistically significant. 

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curve (not shown), is significantly lower in corneas with ectasia.  This suggests that the Brillouin 
parameters, such as slope, modulus and stiffness, are appropriate and sensitive metrics to detect 
ectasia. 
5. In vivo human safety study  
Brillouin technique can be applied to human subjects to measure cornea and lens elasticity 
(10 subjects).  
As part of an ongoing safety study approved by PHC IRB (IRB#2008P002176), we have performed 
the Brillouin imaging procedure on 10 healthy subjects (20 - 60 years old), who have reported no 
discomfort, or complications during or after the imaging session. We have completed the study, 
including the 6-month follow up exam on eight of the study subjects on March 13 2014.  The 
Brillouin test has not caused any ocular problems in this subject. Figures 8a and 8b show 
representative high resolution Brillouin profiles with a pixel integration time of 0.4s of the cornea 
and the lens, respectively. Axial resolution was estimated to be about 60 m enabling local 
measurement of the elasticity of cornea and lens [31]. Lens data also suggests age-related stiffening, 
occurring mainly from the thickening of the stiffer nucleus portion of the lens. Such stiffening has 
long been suspected to be the main cause for the onset of presbyopia, but it had never been measured 
in vivo.  
 
Fig. 8: In vivo Brillouin scan (at 780 nm) of the human cornea (a) and lens (b) 
6. In vivo human study results 
Brillouin measurements are reproducible and sensitive to the targeted corneal changes. 
To calculate the sensitivity of the measurement, we used the in vivo data of the aqueous humor, 
which can be considered as a reference material with constant Brillouin shift; at 0.4s integration 
time, we computed the sensitivity of the instrument to be ~ 30MHz. This is about 10-15 times better 
than the measured difference between normal and ectatic corneas.  
 
Fig. 10: Slope (a) and mean modulus (b) for the human corneas ex vivo (normal, ectasia) and in vivo (healthy individual) 

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To verify the reproducibility and non-perturbative nature of the in vivo measurements, we compared 
four axial scans of the cornea of the same human subject and quantified the variation of slope and 
mean modulus.  We obtained the slope (normalized for comparison with ex vivo) of 0.36 ± 0.14 
GHz/mm and modulus of 2.76 ± 0.015 GPa. The in vivo values are consistent with the ex vivo 
measurements in normal subjects (no statistically significant difference with one-way ANOVA) and 
show a statistically significant difference (unpaired t-test P < 0.01) with the ex vivo ectasia corneas 
(Fig. 10). The measurement is clearly reproducible as the variations from scan to scan are small 
compared to the variation we expect to find due to ectasia.  
C) RATIONALE BEHIND THE RESEARCH, AND THE POTENTIAL BENEFITS TO 
SOCIETY 
The Brillouin microscopy is a safe and non-contact technique that would overcome many of the 
current state of the art technology limitations by providing high resolution 3D spatially-resolved 
information of the elastic modulus of the cornea. 
Significance of Brillouin microscopy for the keratoconus and the LASIK surgery  
This research will introduce a novel diagnostic test for early diagnosis of keratoconus and refractive 
surgery screening that targets the proper biomechanical parameters. As for keratoconus, the novel 
diagnostic tool is expected to enable early identification and better management of disease 
progression as well as development/monitoring of customized treatment via the corneal 
crosslinking.   As for LASIK surgery, this study is expected to drastically change the current status 
quo where many normal eyes are denied the beneficial treatment for vision correction while 
predisposed “weak” corneas are missed and operated upon with terrible consequences. Because of 
this study, early keratoconus patients that are currently not diagnosed, could be recognized and 
treated at a time where the progression of the disease can still be halted. Furthermore, the technique 
will serve as an indispensable tool to monitor and optimize the most promising treatment (currently 
in clinical trials in the US) to stop keratoconus progression, i.e. the corneal collagen crosslinking 
that is based on the stiffening of the corneal stroma and can be easily characterized by the Brillouin 
technology [30]. As for LASIK, because of the validation of the Brillouin technology, subjects with 
weak corneas could be screened before the LASIK surgery and consulted to avoid the operation. In 
addition, since the Brillouin instrument will provide 3D-resolved information on corneal elasticity, 
this research will pose the foundation to develop less invasive, possibly customizable, laser surgical 
procedures based on biomechanical anomalies.  
The broader clinical significance - Several corneal conditions present with altered biomechanical 
properties (e.g. keratoglobus, and pellucidal marginal degeneration). Developing the Brillouin 
microscopy into a viable clinical ophthalmology exam will improve the diagnosis, management and 
treatment of these conditions. In addition, the current assessment of intraocular pressure (IOP) via 
tonometry, crucial for the glaucoma diagnosis and management, is heavily biased by the differences 
in corneal thickness curvature and stiffness among the general population. Only the thickness and 
curvature, however, can be currently accounted for [45]. The Brillouin technique will enable us 
measuring the corneal stiffness, which will improve the accuracy of IOP measurements thus 
providing an improved screening and management of glaucoma patients. Hence, the Brillouin 
instrument validated in this proposal can become a widespread diagnostic device for the corneal 
examinations. 
The feedback from the clinical community - The clinical significance of development of the 
Brillouin microscopy for detection of the corneal ectasia has been recently recognized by the 

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ophthalmology community. Two highly competitive research grants have been awarded for the 
research involved with this pilot study by Harvard Catalyst Advanced Imaging Pilot program and 
by the American Society for Laser Medicine and Surgery.    
The positive medical impact - This research will lead to large-scale clinical studies of Brillouin 
imaging for keratoconus and ectasia patients. If these are successful and the Brillouin imaging is 
demonstrated capable of detecting biomechanical anomalies in patients with early keratoconus and 
post-LASIK ectasia, improved diagnosis and screening protocols will soon follow. In addition, the 
comparison of the biomechanical and topographical features during the disease progression will also 
crucially advance our mechanistic understanding of the keratoconus and ectasia development. 
II. SPECIFIC AIMS 
Our long-term goal is to enable early, reliable identification of individuals at high risk to develop 
keratoconus and ectasia. The objective here is to quantify the biomechanical changes involved in 
keratoconus and in post-LASIK ectasia patients by using Brillouin optical imaging. Our central 
hypothesis is that low Brillouin modulus/stiffness is an early indicator of keratoconus progression 
and a predictor of post-LASIK ectasia occurrence. This hypothesis was formulated on the basis of 
the consensus in the literature regarding the biomechanical origin of ectasia development and our 
preliminary estimation about the mechanical sensitivity of Brillouin optical imaging. The rationale 
for this research is that the collected preliminary data will enable designing two important clinical 
studies focused on biomechanical characterization of keratoconus progression and pre-operative 
elasticity-based screening of LASIK patients. The proposed research consists of three specific aims: 
Specific Aim 1: Compare biomechanical properties of eyes affected by Corneal disease vs. normal 
corneas  
The objective of this aim is to compare the biomechanical Brillouin parameters of keratoconus 
patients, Fuch’s Endothelial Corneal dystrophy patients, and Dry Eye Syndrome patients vs. healthy 
individuals to determine the diagnostic sensitivity of Brillouin measurement. Our working 
hypothesis is that the mechanical degradation of the corneal tissue involved in corneal disease 
progression can be detected by the Brillouin-derived metrics. This hypothesis is supported by 
previous studies ex vivo that have found that human keratoconus corneas to have reduced elastic 
modulus [46-48], an altered collagen organization [49, 50] and reduced number of crosslinks [51], 
and by our previous studies that have shown that the Brillouin imaging is highly sensitive to the 
organizational and crosslinking variations in collagen networks [42, 52]. The data collected here 
will allow us to design clinical studies to test the hypothesis that biomechanical changes develop 
early in keratoconus before irregularities in corneal morphology occur.  
Specific Aim 2: Compare biomechanical properties of post-LASIK ectasia vs. normal corneas   
The objective of this aim is to compare the biomechanical Brillouin parameters of patients who 
developed post-LASIK ectasia and patients who had no complications after LASIK surgery to 
determine the difference in the elasticity between normal post-operative corneas and corneas that 
developed the complication. The working hypothesis of this aim is that the post-LASIK ectasia 
patients will show lower corneal elastic moduli, lower stiffness values, and higher slope levels than 
subjects without complications. This hypothesis is strongly supported by our preliminary data on 
ectasia vs. normal cases ex vivo. The rationale of this aim is that quantifying the difference in corneal 
modulus between post-operative diseased vs. post-operative normal controls, together with the 
measurements of Aim 1, will determine what corneal elasticity levels pre-operatively are to be 
considered at risk. 

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Specific Aim 3: compare biomechanical properties change before and after laser vision correction 
surgeries 
The objective of this aim is to compare the biomechanical Brillouin parameters of partients before 
and after laser vision correction surgeries, i.e., the photorefractive keratectomy (PRK) and LASIK 
surgeries. The working hypothesis of this aim is that post-operative corneas will present lower 
Brillouin stiffness values compared with their pre-operative status, despite Brillouin modulus 
remaining the same. The rational of this aim is that quantifying the difference of Brillouin stiffnes 
between the pre- and post-operative corneas will help us understand the biomechanical property 
changes induced by corneal refractive procedures, and together with aims 1 and 2, will strenghen 
pre-screening to exclude surgery candidates at risk.  
III. SUBJECT SELECTION  
Based on the biostatistical considerations (details are explained in Section VI) estimated upon our 
preliminary data, we have calculated that 168 patients subdivided in 8 groups are necessary and 
sufficient to address the specific aims of this project.  Inclusion/exclusion criteria and source of 
subject recruitment methods are described in this section. 
A) GENERAL INCLUSION/EXCLUSION CRITERIA 
This study includes anyone who has a clear enough cornea or clear enough media to permit imaging, 
and who fulfills all specific inclusion/exclusion criteria of one of the following study groups.  
Excluded from this study are specific volunteers who have occludable narrow angles (without a 
patent peripheral iridotomy). Also monocular volunteers will be excluded. Subjects will be excluded 
if they do not or cannot understand or follow the instructions for the imaging.  For subjects who do 
not speak English, the PHRC policy on “Obtaining and Documenting Informed consent of Subjects 
who do not speak English” will be followed. For signing of the informed consents, volunteers who 
do not speak English will be given a written translation of the short form of the entire English version 
of the consent form in a language understandable to them and will be communicated with through 
a native speaking staff or the interpreter service at MEEI/MGH. 
B) SPECIFIC INCLUSION/EXCLUSION CRITERIA 
Group I: Normal healthy subjects (age 20 - 60, N = 15) 
This group includes healthy subjects with normal appearing corneas respecting all the general 
inclusion/exclusion criteria, with less than ± 3 dioptres refractive correction, normal corneal 
topography (no irregular bow-tie, no skewed axis, no scar or other irregularities), and no history of 
eye diseases, except presbyobia and/or cataract.  
Group II: Keratoconus subjects (age 20 - 60, N = 45) 
This group includes subjects classified as patients with mild, moderate, or advanced keratoconus. 
Inclusion criteria are an irregular cornea determined by distorted keratometry mires, distortion of 
the retinoscopic or ophthalmoscopic red reflex (possibly a combination of the two) and slit-lamp 
biomicroscopic signs such as Vogt’s striae or Fleischer’s ring of over 2 mm arc or corneal scarring 
consistent with keratoconus. The inclusion criteria also extend to forme-fruste keratoconus, or 
subclinical keratoconus, i.e. fellow-eyes of mild, moderate, or advanced keratoconus. The inclusion 
criteria also extend to advanced keratoconus patients scheduled to receive corneal transplant of the 
keratoconic eye. 

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Group III: post-LASIK patients with no complications (age 20 - 60, N = 5) 
This group includes healthy subjects who have undergone LASIK surgery in the past 12 months 
without complications. Inclusion criteria are normal post-operative topography and pachimetry and 
a clinician evaluation of the normal post-operative corneal conditions.  
Group IV: post-LASIK patients with complication of ectasia (age 20 - 60, N = 3) 
This group includes patients who have been diagnosed with post-LASIK ectasia after having 
undergone LASIK surgery more than 12 months prior to our scheduled Brillouin analysis. The 
number of patients in this category is reduced to 3 based on recruiting considerations due to the 
history of reported cases of post-LASIK ectasia at MEEI which may limit the number of eligible 
patients within the projected duration of this study. Our statistical considerations, however, 
demonstrate that imaging 3 patients will allow us to achieve the specified aims. The inclusion 
criterion is the clinical diagnosis of post-LASIK ectasia based on topography, pachymetry and 
clinician evaluation.  
Group V: Patients who are scheduled for PRK surgery (age 20 - 60, N = 10) 
This group includes patients who have been diagnosed with myopia and have been scheulded to 
undergo PRK surgery.  Patients with highastigmatism > 2 diopter, prior ocular surgeries, and those 
patients taking any ocular medications except seasonal allergy medicine such as ketotifen or 
artificial tears will be excluded. 
Group VI: Patients who are scheduled for LASIK surgery (age 20 – 60, N = 10) 
This group includes myopic patients who are scheulded to receive LASIK surgery.  Patients with 
high astigmatism > 2 diopter, prior ocular surgeries, and those paitnets taking any ocular 
medications except seasonal allergy medicine such as ketotifen and artificial tears will be excluded. 
Group VII: Patients who are diagnosed with Fuch’s endotheliul corneal dystrophy (FECD) 
(age 20-75, N = 40). 
This group includes subjects who are diagnosed with Fuch’s corneal dystrophy, at early, mild and 
advanced stages. The inclusion also extends to subjects with, and without keratoconus. But this 
exclude patients with any other corneal disorders other than keratoconus, and/or history of 
ophthalmological surgeries that may affect endothelium cell status, e.g. cataract surgeries. 
Group VIII Patients who are diagnosed with Dry Eye Syndrome (Keratoconjunctivitis Sicca) 
age 20-60, N=40).  
This group includes patients (age 20-60) who are diagnosed with Dry Eye Syndrome 
(Keratoconjuctivitis Sicca). This group included subjects who are diagnosed with Dry Eye 
Syndrome at early, mild, moderate, and advanced stages. The inclusion also extends to subjects with 
and without keratoconus. It excludes patients with any other corneal disorders other than 
keratoconus, and/or history of ophthalmological surgeries that may affect endothelium cell status 
such as previous corneal transplants, cataract surgeries, etc.  
All subjects will be recruited by the Wellman Center for Photomedicine (MGH) through Partners 
HealthCare (PHC) “All Users” email announcements (studies seeking volunteers), ads posted at 
PHC campuses, flyers and classified internet advertisements. Group II and IV subjects will be 

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additionally recruited among Massachusetts Eye and Ear Infirmary (MEEI) patients through the 
procedures described below.   
IV. SUBJECT ENROLLMENT  
1) Volunteers responding to the PHC email announcement or the ads will contact a member of the 
study staff who is available to answer any question regarding the study. To enroll a volunteer, a 
member of the study staff will go through an IRB approved questionnaire on the phone to 
determine whether a volunteer fulfills inclusion/exclusion criteria to be recruited and go on for 
the routine eye exam at Massachusetts Eye and Ear Infirmary (MEEI). The member of the study 
staff will then schedule an appointment for the routine eye exam at MEEI based on the pre-
determined time availabilities agreed among the study staff; otherwise he/she will obtain the 
patients permission to be contacted by the study staff in order to schedule an appointment for 
the routine eye exam at MEEI. The study staff will also ask permission to contact the subject via 
regular or electronic mail to provide: confirmation of the time and date of the appointment; 
address, directions and parking information regarding MEEI; and a copy of the consent form.  
 
2) Subject recruitment can occur from the MEEI or Boston Laser (1101 Beacon Street, Suite 6 
Brookline, MA 02446) patient pool. The study coordinator at MEEI or Boston Laser will access 
medical records, patient registries or clinical databases to identify potential subjects.  To ensure 
that patient privacy is protected and that the individual patient is appropriate to participate in the 
research, the first contact with potential subjects identified through their private health 
information will not be made by the study investigators. Rather, the patient's primary doctor or 
eye specialist will be involved.  
In particular, the patient's primary doctor or eye specialist who has first hand knowledge of the 
patient's medical history must (1) give approval for his/her patient to be contacted for research 
purposes, (2) initially introduce the study to the patient and (3) obtain the patient's permission 
to be contacted by study staff.  
 
Subject recruitment can occur among patients seeing Roberto Pineda (MEEI site PI), Amy Scally 
(co-investigator), 
Matthew Gardiner (co-investigator), Sheila Borboli-Gerogiannis 
(co-
investigator), Amy Watts (co-investigator), Samir Melki (co-investigator), Ula Jurkunas, Joseph 
Ciolino, or Sherleen Chen. In this case, special precautions will be taken to avoid the possibility that 
the patients may feel obligated to participate because they are being asked by their treating 
physician. The study staff and treating physician will make clear that participation is voluntary, that 
patients do not have to participate, and the decision not to participate will not affect their care, now 
or in the future. Further, to minimize the possibility that patients will feel obligated to participate, 
patients will be  
- 
(a) initially contacted about the research study through a recruitment letter, and/or  
- 
(b) Patient’s primary physician will mention the research study to the subjects and give the 
interested subjects the contact info of the study staff so that the subjects initiate the 
enrollment procedure or ask additional questions about the research study; A flyer containing 
an introduction of the study and a picture of the imaging system will be either shown to the 
patients or put up in the physician’s office; and/or  
- 
(c) Patient’s primary physician will ask permission to the subjects for them to be contacted 
by the study staff for further information about the study and possible enrollement; and/or 

Version Date: 09/23/2019 
12 
 
- 
(d) If patients are interested, enrollment and consent procedures will be preferentially 
administered by a member of the study staff rather than the treating physician   
If a patient were to express genuine interest in participating in the study and wanted to enroll into 
the study, the treating physicians may provide the PHC IRB approved consent form to the patient, 
and if the patient consents to the study, they may perform the initial eye exam or provide contact 
info of the study staff to schedule the participation in the study.   
Upon enrollment, each volunteer will be registered as patient at MEEI. A member of the study staff 
will use a PHC IRB approved consent form and one of the co-investigator physicians at MEEI will 
perform the initial eye exam. They will also confirm the eligibility of each volunteer to participate 
in the study. The subject then will then be accompanied by study staff to Wellman Center at MGH 
main campus for the subsequent Brillouin imaging, or will schedule the Brillouin imaging session 
for a later date with a member of the study staff. 
A copy of the signed consent form will be given to the subject to keep for her/his records. 
V. STUDY PROCEDURES  
The study procedures involve an initial eye examination at MEEI to validate the subject eligibility 
plus the study imaging session at Wellman Center (MGH). The detailed description of which is 
provided below.    
A) EYE EXAM AT MEEI  
On appointment day at MEEI, a member of the study staff will explain the study to the volunteer 
and obtain consent at MEEI prior to the eye exam. The standard eye exam may include medical and 
ocular history taking, manifest refraction test, slit lamp biomicroscopy of anterior segment, 
intraocular pressure measurement, and corneal topography (Pentacam). After the Pentacam, a 
member of the study staff will confirm which eye(s) are eligible for the study by marking and signing 
the consent form.  
Before the Brillouin imaging session, the study staff will obtain the signed consent form and a copy 
of the pentacam of the study subject after all the identifying information has been deleted.  
For the Brillouin imaging session, the subject will be escorted by a member of the study staff to 
Wellman Center (MGH); alternatively, the Brillouin imaging session can be scheduled for a later 
date at the convenience of the subject; in this case, an appointment can be taken on that day or the 
study staff can obtain permission to contact the study subject for scheduling purposes. 
If an unexpected condition were to be found during the eye examination, the volunteer would be 
informed and recommended to follow up with her/his regular eye doctor.  If the volunteer does not 
have a regular eye doctor, Dr. Pineda will refer her/him to an appropriate specialist for treatment.  
If treatment for the condition were needed, this would certainly take precedence, and the volunteer 
would not be enrolled in the study. 
New subjects will be recruited instead after amending the protocol for recruiting additional subjects 
for the corresponding group.  
 
 

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B) STUDY IMAGING SESSION AT WELLMAN CENTER (MGH) 
At MGH, the subject undergoes Brillouin imaging on one eye only. The study procedure, Brillouin 
microscopy, will be explained to the subject again when he/she arrives at the Wellman Center, and 
will be given opportunity to ask questions. If the study subject decides to go on with the study, the 
determination of which eye will be imaged will follow.   
Determination of which eye is eligible for Brillouin microscopy examination. Subjects of Group I 
will be given the choice to decide which eye they want to be examined by Brillouin microscopy. 
The subjects, however, will be encouraged to choose the eye they do NOT usually use for monocular 
activities in their daily lives such as taking pictures. Subjects of Group II – VIII will also be given 
the choice to decide which eye they want to be examined by Brillouin microscopy if in the eye 
screening visit both of their eyes were deemed eligible for the study. This will occur if they have 
bilateral keratoconus (Group II), if they had LASIK surgery on both eyes (Group III), if they are 
diagnosed with post-LASIK ectasia in both eyes (Group IV), if they are diagnosed with Fuch’s 
dystrophy in both eyes (Group VII), and if they are diagnosed with Dry Eye Syndrome (Group VIII). 
The subjects can also choose to have both eyes scanned. However, if in the eye screening process, 
only one of the subject’s eyes (corneas) is deemed eligible for our study, the patient will be informed 
that the test could be performed only on the eligible eye. At this time, the subject will have an 
opportunity to consent to the procedure on that eye or withdraw from the study. If the subject 
consents to the procedure, he/she will circle the Left or Right eye segment on the consent form and 
sign the consent form recording the date and time before the imaging procedure.  The MGH co-
investigator, either Dr. Eltony, or Dr. Shao will also confirm the designation of the study eye on the 
consent form, and sign it with date and time as well. If, on his/her own, the patient expresses interest 
in having both eyes examined and both eyes are found eligible for Brillouin microscopy 
examination, then he/she will circle both the Left and Right eye segment on the consent form and 
sign the consent form recording the date and time before the imaging procedure. The MGH co-
investigator, either Dr. Eltony, or Dr. Shao will also confirm the designation of the study eye on the 
consent form, and sign it with date and time as well. 
Brillouin imaging session During the imaging session of the cornea the subject will be asked to sit 
calmly in a chair with his/her chin and forehead on a chin and forehead rest, respectively. These 
subject contact areas will be cleansed with alcohol wipes in-between volunteers. Additionally, the 
subject may be asked to use a “bite bar” during the Brillouin imaging because of its great sensitivity 
to motion in high resolution mode.  A bite bar is a piece of Plexiglas covered with sterile dental-
impression material. The subject will make an impression of his teeth in the bite bar, and rest his 
teeth in this impression for the duration of each scanning to help immobilize his head. The not-to-
image eye will be covered with an eye patch. At operator’s instructions, the subject will fix his/her 
eye on a small red or green blinking light, or onto a projected object such as a Maltese cross. No 
mechanical devices for direct immobilization of the eye will be used. The operator will adjust the 
chin rest, the head position, and the orientation of the Brillouin instrument in order to create the best 
accessible angle to start imaging of the cornea. The localization of the near infrared laser in the eye, 
and the eye gaze angle will be recorded with the white-light bright field microscopy. Then, the 
subject will be asked to hold still while fixing the study eye on the blinking light or projected object 
for up to 5 to 10 s during which the instrument gathers the Brillouin spectrum. The subject will be 
asked to comply with this repeated procedure, 5-50 times per eye to gather in-line depth data in a 
spatially-resolved pattern on the cornea. Each line scan will acquire about 15 to 40 points across the 
corneal depth and will take no longer than 10 s. Short break between the imaging scans will be 
provided to rest and to blink the eye. The entire imaging or scanning session may take up to 60 

Version Date: 09/23/2019 
14 
 
minutes in total.  The subject may ask for breaks or stopping the imaging session at any point. During 
the pause between imaging sessions, the subject will be asked whether they have felt any discomfort 
related to their positioning, with the imaging near infrared light (=780 nm), or with the imaging 
procedure.  
Follow-up Brillouin imaging sessions for patient in Group V and VI Two follow-up Brillouin 
imaging sessions will be conducted at: 1 week, and 4-10 weeks after the surgery for patients in both 
Group V and VI. The imaging sessions will be accomplished after the patients’ follow-up checks at 
MEEI with their physician and the same imaging protocols will be used, as described above. At the 
follow-up checks at MEEI, the patients’ eye of interest will be imaged with both Pentacam, and an 
optical-cohernece tomography imaging system by Dr. Amy Scally to measure the corneal thickness. 
The Brillouin imaging sessions will be subject to the physician’s confirmation of the patient’s 
recovery status and eligibility to continue the study.   
If a volunteer that enrolls in the research study has undergone an eye exam comparable to the one 
that would be given at the beginning of the research study (STEP A) within the past year, the subject 
may elect to waive the initial eye exam (STEP A). In this case, the subject will be asked written 
permission that the file of his previous visit, and in particular his/her topography and pachymetry 
exams, can be reviewed by Dr. Pineda or Amy Scally to confirm eligibility. For the purpose of this 
study, the information needed is a routine eye exam for eligibility purposes; therefore a baseline eye 
exam undergone in a few-month window before the study is sufficient to provide the required 
information.    
If a patient of MEEI or Dr. Pineda’s or Amy Scally’s that enrolls in the research study is scheduled 
to undergo standard check-up examinations at MEEI, as part of his routine eye care,  within a period 
of two years following the imaging session, the subject will be asked permission that the information 
regarding the pachymetry tests or Pentacam can be analyzed by the study staff, in order to correlate 
the Brillouin signatures with the longitudinal evolution of the patient’s cornea topography.   
If a patient expresses interest in a follow-up visit and/or Brillouin examination, they will be 
scheduled at the end of the first visit or the patient will be contacted by the study staff for scheduling. 
A remuneration of $200 will be offered for the completion of the Brillouin imaging study. If the 
subject is determined to be ineligible to participate in the study after the routine eye exam, he/she 
will receive the reimbursement of $25 for the screening exam. The payment by check will be mailed 
to the subject. 
The volunteers will not be charged for the visit or for Brillouin imaging session.  Subjects may 
receive reimbursement for transportation and a parking voucher or $5 reimbursement for MBTA 
tickets. If the subject is coming from more than 200 miles away for the imaging, then we will 
reimburse airfare, meals, and transportation expenses to and from the airport. 
A study subject may contact study PIs for any concerns or questions he/she may have after the study 
procedures are completed. The contact person at the Wellman Center (MGH) is Andy (SH) Yun, 
PhD, Phone # 617-768-8698, and the contact person at the MEEI is Roberto Pineda, MD Phone # 
617-573-3234 or 617-573-4393. 
 

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15 
 
VI. BIOSTATISTICAL ANALYSIS  
A) 
SPECIFIC DATA VARIABLES COLLECTED FOR 
THE STUDY 
Axial scans in different locations of the cornea may be 
performed: one scan in the center, and four in cross pattern at 2-
3 mm distance from the center. In particular, one scan covers the 
region 
where 
keratoconus/ectasia 
is 
most 
prominently 
developing, and another axial scan diametrically 180 opposite 
to it. Axial scanning is performed by electronically moving the 
objective lens with speeds ranging between 30 and 80 μm/s. The 
Brillouin spectra are recorded with frame integration times 
between 0.1 and 1 s. The laser power at the sample will be below 3 mW. The beam scanning, CCD 
acquisition and spectral analysis are synchronized with LABVIEW and MATLAB programs. The 
axial profile of the Brillouin shift is plotted against the corneal depth (Fig. 11) and calculated using 
the refractive index of the anterior chamber measured by the pre-test eye exam (low-coherence 
interferometry).  
 
Elasticity-based metrics derived from Brillouin images.  
Axial analysis: Fig. 11 shows a typical Brillouin axial scan of a cornea and pictorial representation 
of several parameters that can be extracted with their respective definitions.  Mean Brillouin modulus 
is defined as the spatial average (Pa); it can be considered the closest quantity compared to the 
macroscopic modulus measured in traditional mechanical tests. The slope (GHz/mm) is the rate of 
change of modulus across depth. Brillouin stiffness is the integral of the modulus over depth, i.e. the 
area under the curve (Pamm). All the parameters can be calculated for the entire cornea or for a 
specific depth region. Regional variations:  From Brillouin measurements, we also expect to capture 
the biomechanical differences between ectatic and normal areas within the same cornea. These 
measurements represent an ideal intracorneal positive control for our technology assessment. 
B) STATISTICAL METHODS AND POWER ANALYSIS 
 
Specific Aim 1:   Biomechanical comparison of eyes affected by Corneal disease vs. Normal 
corneas      
Hypothesis: Keratoconic corneas have lower (reduced) Brillouin modulus and stiffness compared to 
normal. Fuch’s Endothelial Corneal Dystrophy corneas and Dry Eye Syndrome corneas will exhibit 
different biomechanical properties due to corneal hydration changes.  
Subjects: (Group I: Normal, N = 15); (Group II: Keratoconus, N = 25); (Group VII: Fuch’s 
Endothelial Corneal Dystrophy, N=40); (Group VIII: Dry Eye Syndrome, N=40).  
Analysis Plan: The relationship between normal vs. eyes affected by corneal disease (dichotomous 
explanatory variable) and Brillouin metrics (continuous outcome variable) will be investigated using 
unpaired t-tests.  
Sample size and Power:  Advanced keratoconic corneas have been previously measured elsewhere 
to have a Young’s modulus of 28 ± 4.7 MPa compared to 57 ± 4.1 MPa of normal corneas [47]. 
Using the Young’s-to-longitudinal modulus conversion [42, 52], we expect to observe (acquire) 
Brillouin moduli of 3.57 ± 0.02 GPa and 3.66 ± 0.01 GPa for keratoconic and normal corneas, 
respectively. If the true difference is comparable or greater than this estimation, a sample size of 5 
keratoconus vs. 5 healthy controls allows rejecting the null hypothesis that the population means of 
the experimental and the control groups are equal with the power of 0.9. The Type I error probability 
(p-value) associated with this test equals 0.05. The stiffness parameters that include the thinning 
Fig. 
11: 
Definition 
of 
Brillouin 
elasticity parameters.   
Depth 
Brillouin shift 
slope I 
slope II 
mean
modulus 
Area:
Brillouin stiffness

Version Date: 09/23/2019 
16 
 
effect of keratoconus are expected to have higher separation and thus will have sufficient power and 
type I error probability.  
An additional number of 40 patients is added to establish a correlation between corneal stiffness and 
progression of the disease. As independent variable we will use the K-max obtained from pentacam 
evaluation which varies from ~40D in mild cases to ~70D in advanced keratoconus. As preliminary 
results from this study, a slope of -4 MHz/D and a correlation coefficient of 0.87 was found between 
the minimal Brillouin modulus and K-max (x=13.82D) in moderate to advanced keratoconus. If 
the true slope of the line obtained by regressing Brillouin modulus against K-max is the same for 
mild cases, the use of 40 patients will enable to reject the null hypothesis that this slope equals zero 
with power of 0.8. The Type I error probability associated with this test of this null hypothesis is 
0.05. Furthermore, the additional number of subjects will inform us on the different mechanical 
patterns associated with the progression of keratoconus vs normal corneas.  
 
Specific Aim 2:   Biomechanical comparison of post-LASIK ectasia vs. normal corneas   
Hypothesis: Patients with post-LASIK ectasia will present lower Brillouin modulus/stiffness and 
higher axial slope than post-LASIK patients without complication. 
Subjects: Post-LASIK ectasia, N = 3 (Group III); Post-LASIK no complications, N = 5 (Group III)  
Analysis Plan: The relationship between ectasia vs. normal post-operative conditions (dichotomous 
explanatory variable) and Brillouin parameters (continuous outcome variable) will be investigated 
using unpaired t-tests.  
Sample size and Power: Assuming standard deviation comparable to preliminary data, the sample 
size of 3 post-operative ectasia cases vs. 5 post-operative no-complication controls allows to detect 
a true difference of < 0.4 GHz/mm (slope) and < 0.12 GPa (mean modulus) with 0.05 one-tailed 
alpha and > 80% power. From preliminary data, we estimate the separation between normal and 
ectasia cases to be 0.58 GHz/mm in slope and 0.14 GPa in mean modulus. Therefore, the sample 
size is expected to detect the desired difference with sufficient power and type I error. The stiffness 
parameter has shown higher separations in preliminary data, and it is expected to have sufficient 
power and type I error for this analysis as well. 
 
Specific Aim 3:   Biomechanical comparison of before and after laser vision correction surgeries   
Hypothesis: Patients with post-LASIK ectasia will present lower Brillouin modulus/stiffness and 
higher axial slope than post-LASIK patients without complication. 
Subjects: Patients scheduled to receive PRK surgery, N = 10 (Group V) and LASIK surgery, N = 
10 (Group VI) 
Analysis Plan: The relationship between pre- vs. post-operative conditions (dichotomous 
explanatory variable) and Brillouin parameters (continuous outcome variable) will be investigated 
using paired t-tests.  
Sample size and Power:  
Assuming PRK and LASIK surgeries have similar effects on biomechanical properties of human 
cornea, and that standard deviation comparable to preliminary data, the sample size of 10 patients 
allows to detect a true difference of < 0.03 GPa (mean modulus) with 0.05 alpha and > 90% power. 
This is about 1% of the mean Brillouin modulus of normal cornea according to our preliminary data. 
Other studies reported 10 – 20% change of biomechanical properties in samples received LASIK 
treatment [68]. Therefore, the sample size is expected to detect the desired difference with sufficient 
power and type I error. 
 
 
 

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17 
 
C) 
STUDY ENDPOINTS 
 
1) The studies in Aim 1 will provide biomechanical metrics for keratoconus diagnosis, Fuch’s 
Endothelial Corneal Dystrophy diagnosis, Dry Eye Syndrome diagnosis and progression 
analysis. If the data of Aim 1 show statistically significant differences in Brillouin 
parameters between normal and corneal disease subjects, we will compute the performances 
of a Brillouin diagnostic test for keratoconus, Fuch’s Endothelial Corneal Dystrophy, and 
Dry Eye Syndrome. For this, Brillouin metrics will be treated as continuous predictors of 
dichotomous disease outcome. Receiver operating characteristic (ROC) curves will be traced 
and one- or two-parameter cutoff criteria for optimal sensitivity/specificity will be 
calculated. These criteria will be tested in a future clinical study including subjects with 
subclinical corneal disease (i.e. fellow eyes of patients). The data collected here will also 
provide sufficient information on effect size to design a clinical study aimed at characterizing 
the biomechanical progression of keratoconus, Fuch’s Endothelial Corneal Dystrophy, and 
Dry Eye Syndrome. In addition, the analysis of the regional variation may provide 
preliminary data for a clinical study where Brillouin vs. topographical maps are compared 
to evaluate mechanical and morphological changes in corneal disease. 
2) If the t-test comparison in Aim 2 shows statistically significant differences in Brillouin 
parameters (slope, modulus and stiffness) between the ectasia vs. healthy post-operative 
conditions, we can evaluate the performance of Brillouin microscopy as a diagnostic test for 
post-op ectasia. To do this, we will analyze the same datasets using Brillouin parameters as 
continuous predictors of disease outcome. Assuming the data to be distributed normally, the 
power calculation of the previous section remains valid for the receiver operating 
characteristic (ROC) curves [53]. In addition to the one-parameter ROC curves based on 
single Brillouin metric, we will trace ROC curves employing two parameters, slope plus 
modulus or stiffness. These ROC curves will establish optimal cut-off criteria for maximum 
sensitivity and specificity of the Brillouin test of ectasia in post-operative conditions. To 
translate these results in preoperative conditions and evaluate the optimal cut-off criteria for 
a Brillouin screening test of ectasia, we will account for the “baseline” mechanical change 
of LASIK operations taking into account the measurements in healthy individuals, i.e. 
calculating the reduction of corneal stiffness and modulus that LASIK operation induces in 
normal patients without complications. In this way, we will be able to estimate a lower bound 
of corneal modulus/stiffness that can be considered at risk to develop ectasia pre-operatively.  
VII. RISKS AND DISCOMFORTS 
COMMON: Risks are minimal as the Brillouin Ocular Scanner (BOS) light source power is within 
ANSI and ISO standards, and it is also less than that of the current commercially available systems 
that use similar light sources.  The Brillouin Ocular Scanner has been approved by the radiation 
safety office and biomedical engineering office at Massachusetts General Hospital. In case of any 
emergency, a mechanical shutter will block the near infrared laser beam by the push of an emergency 
button. NO eye dilatation is needed for Brillouin imaging, and discomfort from eye straining due to 
not being able to blink during the scanning. Subjects will be escorted by study staff from MEEI to 
MGH. They will be constantly monitored and asked about their well-being. Risks of infection are 
minimal as the BOS obtains images without direct contact to the eye itself.  Cleansing the chin and 
forehead rests with alcohol wipes in between patients minimizes the risk of infection from subject 
to subject. The dental impression material of the bite bar will be discarded after each imaging 
session. 

Version Date: 09/23/2019 
18 
 
UNCOMMON: We are not aware of any uncommon risks. To ensure compliance with the ANSI 
2007 standards, the light source power used in the Brillouin imaging is measured before each 
imaging session. The values are logged into the experiment book for each imaging session and kept 
on record. If any adjustments are made to the system during a session, the power will be measured 
and recorded again before continuing the imaging session. 
VIII. POTENTIAL BENEFITS  
The volunteers who participate in the study may benefit from the eye examination performed at 
MEEI, which may confirm that the individual has normal eyesight or which may uncover a disorder. 
Should this technology prove effective in measuring corneal elasticity at high sensitivity, there will 
be a potential benefit to the subjects enrolled in the study to gather information about the elasticity 
of their cornea. This can be beneficial if and when decisions have to be made regarding the 
opportunity to undergo refractive surgery. In addition, this could give more accurate Intra-Ocular 
Pressure (IOP) reading by correcting the elasticity bias.  If successful, Brillouin Microscopy may 
become a new gold standard to diagnose the onset and the progression of eye diseases such as 
keratoconus, and may detect at-risk patients for LASIK surgery.   
IX. MONITORING AND QUALITY ASSURANCE  
The laser light exposure in this study is within documented safe limits set by the American National 
Standards Institute (ANSI 2007) and ISO 15004-2:2007. In addition, Brillouin technology operates 
at light exposure levels that are less than that of the current commercially available systems that use 
similar light sources.  
 
The principal investigators will be available to discuss and monitor any changes in the health of the 
subject’s eyes after the imaging sessions. If any problem related to the study imaging session is 
depicted, the study will be immediately altered or stopped, and actions will be taken depending on 
the severity of the injury (see below). All forthcoming appointments will be cancelled by contacting 
the volunteers by phone. The results from the Brillouin imaging study will be monitored bi-weekly 
during a research personnel meeting. A full review of all study procedures and data will be due 
every 3 months. 
 
If during the study or later, the subject wishes to discuss his/her rights as a research subject, his/her 
participation in the study and/or concerns about the study, or a research-related injury, with someone 
not directly involved in the study, or if the volunteer feels under any pressure to enroll in this study 
or to continue to participate in this study, he/she is asked to contact a representative of the Human 
Research Committee at MGH (617) 424-4100, or at MEEI (617) 573-3446.  
 
Any adverse event during the subject’s participation in the study will be documented whether or not 
considered to be related to the study procedures. The following will be included in all safety reports: 
Subject identification number and initials, primary investigator’s name, subject’s date of birth, 
gender, and ethnicity, signs and symptoms and severity, date of onset, date of resolution or death, 
relationship of the event to the study, and all actions taken. All adverse events observed by the co-
investigators, a professional colleague, or the subject himself/herself will be recorded.  
 
Reports of unanticipated problems involving risks to subjects or others will be reported to the 
Partners Human Research Committee in accordance with PHRC unanticipated problems including 
adverse event reporting guidelines through Insight/eIRB within 5 working days/7 calendar days.  In 
case of Adverse Event, a report will be submitted containing a detailed description of the adverse 

Version Date: 09/23/2019 
19 
 
event; the basis for determining that the event is unexpected; the basis for determining that the event 
is possibly related to the research procedures; the basis for determining that the research places 
subjects at an increased risk of harm; and, whether any changes to the research or other corrective 
actions are warranted. In this case, amendments to the protocol to incorporate possible changes 
and/or corrective actions will be considered.  
 
This study is sponsored by Harvard Catalyst | The Harvard Clinical and Translational Science Center 
(National Center for Research Resources and the National Center for Advancing Translational 
Sciences, National Institutes of Health Award #UL1 RR 025758 and financial contributions from 
Harvard University and its affiliated academic health care centers) (Title of the project : ‘Early 
detection of corneal ectasia with in vivo Brillouin optical imaging of corneal biomechanical 
properties’ PI’s: Scarcelli, Yun, and Pineda) and by the American Society for Laser Medicine and 
Surgery (Title of the project : ‘A pilot study to identify “at-risk” subjects for post-LASIK ectasia 
using Brillouin microscopy’, PI: Scarcelli). 
X.  REFERENCES 
[1] 
J. Randleman, "Post-laser in-situ keratomileusis ectasia: current understanding and future 
directions," Current opinion in ophthalmology, vol. 17, pp. 406-418, 2006. 
[2] 
J. H. Krachmer, R. S. Feder, and M. W. Belin, "KERATOCONUS AND RELATED 
NONINFLAMMATORY 
CORNEAL 
THINNING 
DISORDERS," 
Survey 
of 
Ophthalmology, vol. 28, pp. 293-322, 1984 1984. 
[3] 
J. O. Jimenez, J. C. G. Jurado, F. J. B. Rodrigues, and D. S. Laborda, "Keratoconus: Age of 
onset and natural history," Optometry and Vision Science, vol. 74, pp. 147-151, Mar 1997. 
[4] 
G. Wollensak, "Crosslinking treatment of progressive keratoconus: new hope," Current 
Opinion in Ophthalmology, vol. 17, pp. 356-360, Aug 2006. 
[5] 
D. Dawson, J. Randleman, H. Grossniklaus, T. O'Brien, S. Dubovy, I. Schmack, R. Stulting, 
and H. Edelhauser, "Corneal ectasia after excimer laser keratorefractive surgery: 
histopathology, ultrastructure, and pathophysiology," Ophthalmology, vol. 115, pp. 2181-
24091, 2008. 
[6] 
Y. Rabinowitz, "Ectasia after laser in situ keratomileusis," Current opinion in 
ophthalmology, vol. 17, pp. 421-427, 2006. 
[7] 
U. de Sanctis, C. Loiacono, L. Richiardi, D. Turco, B. Mutani, and F. M. Grignolo, 
"Sensitivity and specificity of posterior corneal elevation measured by Pentacam in 
discriminating keratoconus/subclinical keratoconus," Ophthalmology, vol. 115, pp. 1534-
1539, Sep 2008. 
[8] 
X. H. Li, Y. S. Rabinowitz, K. Rasheed, and H. Y. Yang, "Longitudinal study of the normal 
eyes in unilateral keratoconus patients," Ophthalmology, vol. 111, pp. 440-446, Mar 2004. 
[9] 
I. G. Pallikaris, G. D. Kymionis, and N. I. Astyrakakis, "Corneal   ectasia   induced   by laser   
in   situ   keratomileusis.," J   Cataract   Refract   Surg, vol. 27, pp. 1796-1802, 2001. 
[10] 
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