\set{final}

\def\Author{Ziebarth}
\def\author{ziebarth}
\def\vol{13}
\def\year{2007}
\def\anum{54}
\def\pages{504-510}
\def\txt_title{Atomic force microscopy measurements of lens elasticity in monkey eyes}
\def\txt_authors{Noel M. Ziebarth, Ewa P. Wojcikiewicz, Fabrice Manns, Vincent T. Moy, Jean-Marie Parel}

\def\rcvd{28 September 2006}
\def\accept{29 January 2007}
\def\publ{2 April 2007}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links

\| Internal defs
\def\Noel{No\euml l}


\article{


\title{Atomic force microscopy measurements of lens elasticity in monkey
eyes}


\authors{\mailto{nziebarth@med.miami.edu}{\Noel\ M. Ziebarth},\sup{1,2}
\mailto{ewawojci@chroma.med.miami.edu}{Ewa P. Wojcikiewicz},\sup{3}
\mailto{fmanns@miami.edu}{Fabrice Manns},\sup{1,2}
\mailto{vmoy@miami.edu}{Vincent T. Moy},\sup{3}
\mailto{jmparel@med.miami.edu}{Jean-Marie Parel}\sup{1,2,4,5}}

\institutions{\sup{1}The Ophthalmic Biophysics Center, Bascom Palmer Eye
Institute, University of Miami Miller School of Medicine, Miami, FL;
\sup{2}Biomedical Optics and Laser Laboratory, Department of Biomedical
Engineering, University of Miami College of Engineering, Coral Gables,
FL; \sup{3}Department of Physiology and Biophysics, University of Miami
Miller School of Medicine, Miami, FL; \sup{4}University of Liege
Department of Ophthalmology, CHU Sart-Tillman, Liege, Belgium;
\sup{5}Vision CRC, University of New South Wales, Sydney, Australia}

\correspondence{Jean-Marie Parel, Ph.D., Bascom Palmer Eye Institute,
1638 NW 10th Avenue, Miami, FL, 33136; Phone: (305) 326-6369; FAX: (305)
326-6139; email: jmparel@med.miami.edu}

\abstract

\abs_purpose{To demonstrate the feasibility of measuring the elasticity
of intact crystalline lenses using atomic force microscopy (AFM).}

\abs_methods{AFM elasticity measurements were performed on intact lenses
from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, \lt 1 day
postmortem) that had been left attached to their zonule-ciliary
body-sclera framework. The eyes were prepared by bonding a plastic ring
on the sclera after removal of the conjunctival, adipose, and muscle
tissues. The posterior pole was sectioned, with the excess vitreous
removed, and the eye's anterior section was placed on a Teflon slide to
protect the posterior pole of the lens. The cornea and iris were then
sectioned. The lens-zonule-ciliary body-sclera section was then placed
in a Petri dish filled with balanced salt solution in an AFM system
designed for force measurements. Next, the central pole of the anterior
surface of the intact lens was probed with the AFM cantilever tip. The
recorded AFM cantilever deflection-indentation curves were used to
derive force-indentation curves for the lens after factoring out the
deflection of the cantilever on a hard surface. Young's modulus of the
lens was calculated from the force-indentation relation using the Hertz
model.}

\abs_results{Young's modulus was 1,720\pom 880 Pa (range: 409-3,210 Pa)
in the 18 cynomolgus monkey lenses.}

\abs_conclusions{AFM can be used to provide measurements of the
elasticity of the whole lens including the capsule. Values obtained
using AFM on cynomolgus monkey lenses are similar to published values
obtained using dynamic mechanical analysis on young human lenses.}

\introduction

\p{Presbyopia is the progressive loss of accommodation with age [1-4].
Even though the exact causes of presbyopia are still not fully
understood, it is generally believed that its origins are multifactorial
and involve several of the accommodative structures, including the lens,
ciliary muscle, ciliary body, and zonules. A number of studies suggest
that presbyopia entails a loss of lens elasticity with age [5-8].}

\p{The elasticity of the lens has been previously investigated using a
spinning method [5], uniaxial stretching [9], squeezing [6], and dynamic
mechanical analysis [7,8,10]. The spinning method, uniaxial stretching,
and squeezing provided relative values of lens elasticity that could be
used to study age-related changes but did not provide absolute values
needed for mechanical models. Dynamic mechanical analysis (DMA) provides
absolute values of lens elasticity but it requires special attention
during the calibration of the device and the preparation of the tissue.
Heys et al. [7] performed measurements on human lenses that were frozen
at -80 \deg C, partially thawed, sectioned equatorially, and then cored
using an 8.5 mm internal diameter trephine. Measurements were performed
at 22 \deg C, and dehydration was prevented using moistened foam rubber
surrounding the probe. Weeber et al. [8] performed measurements on human
lenses that were frozen at -70 \deg C, defrosted, and then sectioned.
Measurements were performed at 36 \deg C, and dehydration was prevented
using silicone oil. Published values of lens elasticity measured using
DMA differed by several orders of magnitude in older lenses, most likely
due to these differences in methodology [7,8].}

\p{Another technique that could provide insight into lens elasticity and
changes with age is atomic force microscopy (AFM). In medicine and
biology, AFM has been used previously for elasticity measurements of
individual cells [11-16], proteins [17,18], and soft tissue [19]. The
purpose of the present study was to demonstrate the feasibility of using
AFM to measure local in situ lens elasticity in a manner that is
atraumatic to the tissue.}

\methods

\subsection{Atomic force microscope}

\p{The AFM system used was a laboratory-made modification of the AFM
design used for imaging [20,21] (\figref{1}). It was shielded inside an
acoustic/vibration isolation chamber. The AFM cantilever tip (60 nm gold
coating, 0.2 mm tip, MLCT-AUHW, Veeco, Santa Barbara, CA) was lowered
onto the sample at a rate of 5 mm/s. A piezoelectric mechanism (Physik
Instrumente, Karlsruhe/Palmbach, Germany) moved the cantilever
vertically in response to applied voltage. During the elasticity
measurements, the cantilever was lowered onto the sample and underwent
bending following contact with the sample. The degree of this bending
was related to the mechanical properties of the sample: the harder the
sample, the more the cantilever bent. The beam of a diode laser was
reflected off the cantilever surface and underwent deflection in
response to the cantilever bending. The cantilever deflections were
monitored by a position-sensitive two segment photodiode (UDT Sensors,
Hawthorne, CA). Custom software controlled the piezoelectric translator
and timing of the measurements.}

\subsection{Calibration of the atomic force microscope cantilever}

\p{Each AFM cantilever was calibrated before an experiment to determine
its spring constant [22]. This was accomplished by first recording the
voltage detected at the photodiode due to deflection of the cantilever
as a function of piezoelectric displacement to determine the
relationship between voltage and cantilever deflection. This scan was
conducted by placing the cantilever in contact with the bottom of a
Petri dish with BSS with an indentation force of 1 nN (\figref{2}{A}).
To determine the thermally induced fluctuation of the cantilever, we
lowered the cantilever tip so that it was submerged in BSS, but not
touching the bottom of the Petri dish (\figref{2}{B}). Equation 1 shows
the measured variance of the deflection used to calculate the spring
constant:}

\ctr{\gifimage{9}{300}{199}{8}}

\p{where \i{k\sub{B}} is Boltzmann's constant, \i{T} is temperature, and
\lt\i{\Chi\sup{2}}\gt\ is the variance of the cantilever deflection. The
spring constants obtained using this methodology were consistent with
the nominal value of 10 mN/m given by the manufacturer (Veeco).}

\subsection{Experimental protocol}

\p{AFM measurements were made on the central anterior surface of 18
intact lenses (six pairs, six unpaired) from healthy cynomolgus monkeys
(\i{Macaca Fascicularis}, 7.2\pom 2.1 years, range: 4.2-10 years).
Enucleated monkey eyes were obtained from the University of Miami
Division of Veterinary Resources following approved institutional animal
care guidelines through an approved tissue-sharing protocol. Eyes were
obtained from monkeys euthanized for experiments not related to the
current study. After enucleation, all eyes were placed in sealed
containers with gauze soaked with BSS to prevent dehydration of the
globe. All eyes were stored at 5 \deg C and returned to room temperature
before they were used. Experiments were performed no more than one day
postmortem (0.25\pom 0.27days).}

\p{The protocol described as follows was used to prepare the lens for
AFM measurements. A custom-made circular black plastic ring was machined
to fit the average globe radius of curvature (9.5 mm). The ring was
bonded onto the sclera in the region of the ciliary body approximately 2
mm posterior to the limbus using cyanoacrylate adhesive (Duro Quick Gel
super glue, Loctite Corp., Rocky Hill, CT) after the conjunctival,
adipose, and muscle tissues were removed. The ring enabled dissection of
the globe with minimal deformation while keeping the ciliary
body-zonule-lens framework intact. Dissection was not initiated until
the glue had dried. This was to ensure that the glue fumes did not cause
any surface dehydration of the lens. The posterior pole was removed by
making a circumferential incision through the sclera. Excess vitreous
was carefully removed, and the eye section was placed on a Teflon slide.
The cornea and iris were then sectioned. The clinical appearance of each
lens was examined under the operation microscope. All lenses were noted
to be intact and clear. The posterior pole of the lens remained intact
in all eyes. The mounted tissue specimen was then placed in a Petri dish
filled with BSS under the polymethylmethacrylate block containing the
AFM cantilever (\figref{3}). The lens was positioned visually so that
the cantilever tip was over the central pole of the anterior surface.
The tip was lowered until it just touched the surface of the lens. This
position was determined by the point when the reflected laser beam
moves. The tip was then lowered, using the piezoelectric control, so
that it was in a position to probe the surface of the lens. The
measurements were conducted using an indentation force of 300 pN, 0.25 s
of contact time with the sample, and a cantilever retraction speed of 5
mm/s. The voltage detected at the photodiode due to deflection of the
cantilever was recorded as a function of piezoelectric displacement.
These recordings were repeated approximately 10 times per lens. The lens
positioning technique was validated by measuring one cynomolgus monkey
intact lens in five different locations around the center (\figref{4}).
The same protocol was used to demonstrate that the AFM could provide
reliable measurements on a sample with a shape similar to that of the
lens and mounted with the same approach. This was done by measuring a
silicone intraocular lens of known modulus of elasticity.}

\subsection{Data analysis}

\p{The lens indentation was calculated by subtracting the piezo
displacement when probing the sample from the piezo displacement when
probing the hard Petri dish. Force was calculated from the spring
constant (k\sub{C}, mN/m) and slope of the cantilever deflection versus
piezo displacement relationship (C, m/V) found during calibration using
the following equation:}

\ctr{\gifimage{10}{301}{84}{4}}

\p{where \Delta\i{V} is the change in voltage (\i{V}) recorded during
the scans (\figref{5}). The elastic modulus was found using the Hertz
Model [23]:}

\ctr{\gifimage{11}{500}{158}{8}}

\p{where \i{F} is the measured force (\i{N}), \i{K} is Young's modulus
(Pa), \i{v} is Poisson's ratio (\i{v}=0.5), \theta\ is the angle of
indentation (normally assumed to be 55\deg), and \alpha\ is the measured
indentation (m). The determination of Young's modulus was carried out by
least square analysis of the measured force-indentation curves using
data analysis software (OriginLab Corporation, Northampton, MA). Each
curve fit was verified visually. The average of the values was then used
as the modulus for that sample.}

\results

\p{All values of Young's modulus recorded (\tabref{1}) at the different
positions around the center of the lens were between 6% and 35% of the
values recorded at Position 1.}

\p{AFM measurements were performed on a silicone intraocular lens with a
Young's modulus of 3.55 MPa (determined by dynamic mechanical analysis).
Our AFM system showed the modulus was 3.87 MPa. This is a percent
difference of 9%.}

\p{Young's modulus was 1,720\pom 882 Pa (range: 409-3,210 Pa) in the 18
cynomolgus lenses (\tabref{2}).}

\discussion

\p{This study demonstrated the feasibility of using AFM for whole lens
elasticity measurements in situ. For 18 cynomolgus eyes, we found an
elastic modulus between 409 and 3,210 Pa. Since the AFM tip indents the
lens capsule, we initially thought that the values obtained would
correspond to the elasticity of the lens capsule alone. Previous studies
[24-26] showed the lens capsule had an elastic modulus ranging from 0.3
to 6 MPa. These values were three orders of magnitude greater than the
values found in the current study. This large difference suggested that
the AFM measured the Young's modulus of the entire anterior portion of
the lens, including the capsule, epithelial cells, and cortex. This
indicated that the capsule became deformed in bending mode under the
pressure of the AFM tip, which required much less force than deforming
the lens matter in compression mode. If this hypothesis is correct, then
the force exerted on the sample by the AFM tip indented only the softest
part of the lens: the cortex. However, it is likely that the presence of
the lens capsule influenced the results.}

\p{The results obtained on the intraocular lens confirmed that the AFM
provided accurate values of elasticity on samples with a shape similar
to that of the lens and that the modulus of elasticity corresponded to
the bulk modulus of elasticity of the sample. There are currently no
intraocular lenses or commercial lenses available with an elastic
modulus similar to that of the human crystalline lens. The modulus of
elasticity of the intraocular lens is approximately three orders of
magnitude greater than that of the crystalline lens. The conclusions
from the experiments on the intraocular lens can therefore not
necessarily be applied to the crystalline lens. Additional studies on
more suitable models of the lens are needed to confirm these findings.}

\p{For this feasibility study, AFM elasticity measurements were
performed on cadaver cynomolgus monkey lenses. Monkey lenses were used
because they were readily available to our laboratory through
tissue-sharing protocols from other research studies. The monkey eyes
were received immediately after euthanasia, which ensured that
measurements were obtained on fresh tissue. This eliminated potential
measurement artifacts due to lens swelling and water uptake that can
occur in human eyes obtained from eye banks because of storage
conditions and increased postmortem time [27]. Leaving the lens in the
eye does permit ion and water entry into the lens. However, our protocol
was based on previous studies on lens preservation [27], which showed
that there is no water uptake in fresh monkey lenses. The immersion in
DMEM will prevent or reverse this effect if the postmortem time is
short, as it was in this study.}

\p{To the best of our knowledge, the only data available on the elastic
properties of the lens have been obtained on human tissue. Monkeys have
a lens structure, composition, and accommodative mechanism that is
qualitatively similar to that of the human [28-35]. The lens elasticity
in prepresbyopic monkeys and humans should therefore be similar. Heys et
al. [7] provided values of the shear modulus of elasticity (G) and
Weeber et al. [8] provided values for the compliance (1/G) for the
cortex of human lenses. For comparison, these values were converted to
Young's modulus (\i{E}) using the following relationship:
\i{E}=2\i{G}(1+\nu), where \nu\ is the Poisson ratio (assumed to be
0.5). Our values were comparable to those obtained by Heys et al. [7]
and Weeber et al. [8] for the cortex of prepresbyopic human lenses
(98.3\pom 64.5 Pa less than 30 years old [7]; 530-5,300 Pa less than 40
years old [8]; \tabref{3}). These findings suggest that AFM provides
values similar to those obtained using DMA despite differences in the
approach and potential differences between human and monkey lens
elasticity. The values obtained with AFM on monkey eyes are within the
range that would be expected under the assumption that human and monkey
lenses have similar properties.}

\p{Ten successive measurements were performed on each lens and then
averaged to provide Young's modulus for that lens. The variability of
repeated measurements of the same lens (standard deviation divided by
average) ranged from 4.5% to 40.5%, with an average of 17.7% for
cynomolgus monkey eyes. This variability is within the same range of the
variability of the DMA results. Heys et al. [7] reported a variability
of up to 30%, and Weeber et al. [8] reported a variability average of
6%. A number of factors could affect the variability of the
measurements, including changes in hydration as well as inherent changes
in the tissue postmortem.}

\p{From the experiment measuring Young's modulus of the same lens in
different locations around the center, we found differences up to 35%
due solely to change in position. This effect of position on Young's
modulus most likely contributed to the differences we encountered with
paired eyes as well as between animal variability. However, this 35%
variance does not account for the large range of values obtained. The
measured elastic modulus varies by approximately eight times between the
smallest and largest value. The origin of this large variation remains
to be investigated. In separate experiments, we found that a large
variability in the biometric and optical parameters of cynomolgus monkey
eyes obtained from the same source. This variability is also consistent
with the results of Weeber et al. [8] on human lenses. They found that
the mechanical properties of eyes from different donors of the same age
deviated by at least one order of magnitude. This could indicate that
the lens mechanical properties have a relatively high variability
between donors of the same age. However, it is more likely that the
variations are introduced by the tissue preparation, storage, and
handling, independent of the measurement technique.}

\p{In summary, our results demonstrate the feasibility of using AFM to
measure the elasticity of the whole lens including the capsule with
minimal disruption of the tissue. The values obtained on cynomolgus
monkey lenses are comparable to those obtained using dynamic mechanical
analysis on human lenses. Additional studies are under way to quantify
the respective contribution of the lens capsule, cortex, and nucleus to
the measured modulus of elasticity and quantify the effect of age.}

\acknowledgements

\p{We are grateful to the Diabetic Research Institute of the University
of Miami for providing the tissues. We thank the following for their
support of this research: NSF Graduate Student Fellowship; NIH EY14225;
Florida Lions Eye Bank; NSF-BITC; NIH GM55611; the Australian government
through its Cooperative Research Centre Program; NIH center grant
P30-EY014801; Research to Prevent Blindness; and Henri and Flore Lesieur
Foundation.}

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}

\beginfigures

\figfile{1}{
\figtitle{1}{Atomic force microscope for elasticity
measurements}

\p{The atomic force microscopy (AFM) system for elasticity measurements
is a laboratory-made modification of the AFM design used for imaging
[15,20]. It is shielded inside an acoustic/vibration isolation chamber.
\panel{A}: The cantilever is moved vertically using a piezoelectric
translator that responds to applied voltage. A Petri dish containing the
lens is placed below the cantilever, and the cantilever is lowered onto
the lens. The cantilever is bent, causing the beam of the laser diode to
be deflected. A photodiode monitors these deflections. Custom software
controls the piezoelectric translator and times the measurements.
\panel{B}: Shown is a labeled photograph of the AFM used for lens
capsule elasticity measurements.}

\ctr{\gifimage{1a}{600}{554}{46}}

\ctr{\jpgimage{1b}{600}{703}{82}}

}

\figfile{2}{
\figtitle{2}{Calibration scans performed to characterize the atomic force microscope cantilever}

\p{\panel{A}: Force scan conducted on a hard surface to determine
voltage-deflection relationship. A force scan was conducted on the
surface of the Petri dish with no sample to determine the relationship
between voltage detected at the photodiode and cantilever deflection.
The curve provides the voltage-displacement curve in the absence of
indentation. This response is used in the calculation of the
force-indentation curves obtained on a samples. \panel{B}: Recording of
natural vibrational frequency of cantilever in fluid. The thermally
induced cantilever fluctuations are measured by recording the
vibrational frequency of the cantilever in fluid. This response is used
to measure the spring constant using equation 1.}

\ctr{\gifimage{2}{600}{807}{98}}

}

\figfile{3}{
\figtitle{3}{Location of the lens sample in the atomic force microscopy system}

\p{Location of the lens sample in the atomic force microscopy system.
The lens sample is placed in a Petri dish filled with DMEM. The dish
with the lens is then placed in the AFM system under a PMMA block that
contains the AFM cantilever.}

\ctr{\jpgimage{3}{600}{449}{79}}

}

\figfile{4}{
\figtitle{4}{Measurement locations used to
validate the visual lens positioning technique}

\p{Measurement locations used to validate the visual lens positioning
technique. The same lens was measured in five different locations around
the lens center. These measurements were used to validate the sample
positioning technique and to determine the effect of probe positioning
on the variability of the measurements.}

\ctr{\gifimage{4}{600}{598}{88}}

}

\figfile{5}{
\figtitle{5}{Analysis process for atomic force microscopy measurements}

\p{Force scans are taken by probing the sample with the cantilever and
recording the cantilever deflection (upper panel). Force (in
picoNewtons) versus indentation was derived from the cantilever spring
constant and slope found during calibration (lower panel). Young's
modulus was then calculated using the Hertz model.}

\ctr{\gifimage{5}{600}{897}{67}}

}

\begintables

\tabfile{1}{
\tabtitle{1}{Young's modulus obtained at five different locations around the
center of an intact cynomolgus monkey lens}

\p{Young's modulus obtained at five different locations around the
center of an intact cynomolgus monkey lens. Young's modulus was measured
in the center and at four additional locations 1mm from the center for
one cynomolgus monkey lens (see \figref{4}). The values at the different
locations were within 35% of the original measurement at the center.}

\ctr{\gifimage{6}{800}{174}{28}}

}

\tabfile{2}{
\tabtitle{2}{Young's modulus of elasticity obtained for 18 cynomolgus
monkeys}

\p{Each lens measurement was repeated 10 times successively. The
variability was defined as standard deviation divided by the average
multiplied by 100%.}

\ctr{\gifimage{7}{728}{829}{33}}

}

\tabfile{3}{
\tabtitle{3}{Summary of lens elasticity measurements}

\p{The values provided for Fisher correspond to the polar elasticity of
the whole lens, whereas the values provided for Heys et al. [7] and
Weeber et al. [8] are for the lens cortex only.}

\ctr{\gifimage{8}{800}{370}{43}}

}