Supported by National Institutes of Health grant R01 EY035278 (MR).

Pig eyes were obtained from a local abattoir. No ethical committee approvals were required.
1. Lens dissection (Figure 1)
<ol>
	<li>Remove all the surrounding tissue from the pig eyes and excess flesh from the sclera, until only the optic nerve remains. Use curved forceps and small dissection scissors to complete this process. Use the nerve as an anchor to hold the eye during dissection.</li>
	<li>Using a scalpel, make a short circumferential cut at the limbus, then another at the equator. 
	NOTE: This step is performed in this order to avoid damaging the lens and capsule.</li>
	<li>Insert microscissors into the cut at the limbus and remove the cornea by lifting the cornea with fine blunt-tipped forceps while cutting around the circumference of the cornea.</li>
	<li>Remove the iris by lifting using blunt-tipped forceps and cut away with micro-scissors.</li>
	<li>Insert dissection scissors into the equatorial cut, then cut circumferentially around the entire equator until the sclera is bisected.</li>
	<li>Once the cut is completed, remove the posterior portion of the sclera. Remove the vitreous gently with forceps, leaving minimal remnants to avoid damaging the lens. If required, cut the vitreous humor coronally to allow the posterior to pull away from the lens and anterior segment.</li>
	<li>Make a meridional cut through the sclera from anterior to posterior using microscissors.</li>
	<li>Beginning at the new meridional cut through the sclera, use microscissors to cut the zonules away from the lens. Using the weight of the lens or edge of the dissection dish, gently stretch the zonules when pulling the lens and sclera apart slightly, allowing the microscissors to cut between the lens and ciliary body, through the zonules, and around the circumference of the lens. This will isolate the lens without damaging the lens capsule if done correctly.</li>
	<li>If desired, remove the capsule using forceps to puncture the capsule at its equator, then peel away the capsule using two forceps.</li>
	<li>Place the lens in phosphate-buffered saline (PBS). Visually inspect the lens for any damage prior to mechanical testing.</li>
</ol>
2. Lens compression-with/without lens capsule (Figure 2)
NOTE: All steps here with the exception of steps 2.1 and 2.4 are computer-controlled.
<ol>
	<li>Obtain or construct a parallel plate compression apparatus having a 50 gram-force capacity load cell with the ability to measure displacement on the order of 1 &#181;m.</li>
	<li>Program the motorized stage and load the cell to perform the loading regimen described below (e.g., Supplemental File 1).</li>
	<li>Nearly fill a square box of 1 5/8 inches x 1 5/8 inches with PBS and place it on the compression platform.</li>
	<li>Lower the upper plate into contact with the lower plate to determine the lower limit of motion and absolute gap height.</li>
	<li>Raise the upper plate by ~15 mm.</li>
	<li>Center the lens in the box, taking care that the equatorial plane is horizontal.</li>
	<li>Lower the upper plate close to, but not in contact with, the upper surface of the lens.</li>
	<li>Initiate motion to move the upper plate into contact with the lens, using force feedback with a contact threshold of 3 mN.</li>
	<li>Commence data recording upon determination of contact, recording time, position of the upper plate relative to the lower plate, and force at 500 Hz.</li>
	<li>Apply a preconditioning loading where the lens is compressed by 2.5% of its initial height three times, then 5% three times, then 7.5% three times at a rate of 1%/s.</li>
	<li>Hold the position of the upper plate constant for 1 min after preconditioning.</li>
	<li>Apply a 15% compression at a rate of 1%/s, followed by unloading at the same rate.</li>
	<li>Continue the unloading motion until the upper plate has traveled an additional 2% of the unloaded lens thickness away from the bottom plate to ensure the lens is de-adhered from the upper plate.</li>
</ol>
3. Estimation of lens modulus
<ol>
	<li>Estimate the thickness of the lens based on the instrument&#39;s gap at the point of contact. Alternatively, use image analysis to measure the thickness from a photograph taken before testing.</li>
	<li>Compute the elastic modulus E using the Hertz model for compression of a sphere between parallel plates (equation [1]; Supplemental File 2). 
	<img alt="Equation 1" src="/files/ftp_upload/66040/66040eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
	Where R is the radius of curvature at the point of contact (assumed equal to half the lens thickness); F is the compression force reported by the load cell; <img alt="Equation 2" src="/files/ftp_upload/66040/66040eq02.jpg" style="margin: auto;" /> is the Poisson&#39;s ratio (assumed equal to 0.5 corresponding to an incompressible material); and u is the downward approach of the upper stage from the point of contact. Note that the elastic modulus and Poisson&#39;s ratio are material properties indicating respectively the intrinsic stiffness of the lens and the lens&#39; relative compressibility. 
	NOTE: This method neglects any role of the lens capsule but does approximately account for the size of the lens, allowing comparison between species.</li>
</ol>

Biomechanical Characterization of Porcine Ocular Lens with Automated Compression

<ol>
	<li>Gullstrand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helmholtz's+treatise+on+physiological+optics.+translated+edn.">Helmholtz's treatise on physiological optics. translated edn.</a> The Optical Society of America. , (1924).</li><li>Helmholtz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+akkommodation+des+auges.">Uber die akkommodation des auges.</a> Arch Ophthalmol. 1, 1-74 (1855).</li><li>Burd, H. J., Wilde, G. S., Judge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+spinning+lens+test+to+determine+the+stiffness+of+the+human+lens.">An improved spinning lens test to determine the stiffness of the human lens.</a> Exp Eye Res. 92 (1), 28-39 (2011).</li><li>Reilly, M. A., Martius, P., Kumar, S., Burd, H. J., Stachs, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+response+of+the+porcine+lens+to+a+spinning+test.">The mechanical response of the porcine lens to a spinning test.</a> Z Med Phys. 26 (2), 127-135 (2016).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastic+constants+of+the+human+lens.">The elastic constants of the human lens.</a> J Physiol. 212 (1), 147-180 (1971).</li><li>Erpelding, T. N., Hollman, K. W., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+mapping+the+elastic+properties+of+the+lens+using+bubble-based+acoustic+radiation+force.">Spatially mapping the elastic properties of the lens using bubble-based acoustic radiation force.</a> IEEE Ultrasonics Symp. 1, 613-616 (2005).</li><li>Erpelding, T. N., Hollman, K. W., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+age-related+elasticity+changes+in+porcine+lenses+using+bubble-based+acoustic+radiation+force.">Mapping age-related elasticity changes in porcine lenses using bubble-based acoustic radiation force.</a> Exp Eye Res. 84 (2), 332-341 (2007).</li><li>Yoon, S., Aglyamov, S., Karpiouk, A., Emelianov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+pulse+repetition+frequency+ultrasound+system+for+the+ex+vivo+measurement+of+mechanical+properties+of+crystalline+lenses+with+laser-induced+microbubbles+interrogated+by+acoustic+radiation+force.">A high pulse repetition frequency ultrasound system for the ex vivo measurement of mechanical properties of crystalline lenses with laser-induced microbubbles interrogated by acoustic radiation force.</a> Phys Med Biol. 57 (15), 4871-4884 (2012).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li><li>Weeber, H. A., Gabriele, E., Wolfgang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness+gradient+in+the+crystalline+lens.">Stiffness gradient in the crystalline lens.</a> Graefes Arch Clin Exp Ophthalmol. 245 (9), 1357-1366 (2007).</li><li>Reilly, M. A., Ravi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microindentation+of+the+young+porcine+ocular+lens.">Microindentation of the young porcine ocular lens.</a> J Biomech Eng. 131 (4), 044502 (2009).</li><li>Gu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+50+and+AQP0+are+essential+in+maintaining+organization+and+integrity+of+lens+fibers.">Connexin 50 and AQP0 are essential in maintaining organization and integrity of lens fibers.</a> Invest Ophthalmol Vis Sci. 60 (12), 4021-4032 (2019).</li><li>Sharma, P. K., Busscher, H. J., Terwee, T., Koopmans, S. A., van Kooten, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+the+viscoelastic+properties+of+human+and+animal+lenses.">A comparative study on the viscoelastic properties of human and animal lenses.</a> Exp Eye Res. 93 (5), 681-688 (2011).</li><li>Cheng, C., Gokhin, D. S., Nowak, R. B., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+application+of+glass+coverslips+to+assess+the+compressive+stiffness+of+the+mouse+lens:+strain+and+morphometric+analyses.">Sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses.</a> J Vis Exp. (111), e53986 (2016).</li><li>Baradia, H., Negin, N., Adrian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lens+stiffness+measurements.">Mouse lens stiffness measurements.</a> Exp Eye Res. 91 (2), 300-307 (2010).</li><li>Reilly, M. A., Cleaver, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+elastographic+method+for+analyzing+the+ocular+lens+compression+test.">Inverse elastographic method for analyzing the ocular lens compression test.</a> J Innov Opt Health Sci. 10 (06), 1742009 (2017).</li><li>Hahn, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+ex+vivo+porcine+lens+shape+during+simulated+accommodation,+before+and+after+fs-laser+treatment.">Measurement of ex vivo porcine lens shape during simulated accommodation, before and after fs-laser treatment.</a> Invest Ophthalmol Vis Sci. 56 (9), 5332-5343 (2015).</li><li>Parreno, J., Cheng, C., Nowak, R. B., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+strain+on+mouse+eye+lens+capsule+and+cellular+microstructure.">The effects of mechanical strain on mouse eye lens capsule and cellular microstructure.</a> Mol Biol Cell. 29 (16), 1963-1974 (2018).</li><li>Yoon, S., Aglyamov, S., Karpiouk, A., Emelianov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+ex+vivo+bovine+and+porcine+crystalline+lenses:+age-related+changes+and+location-dependent+variations.">The mechanical properties of ex vivo bovine and porcine crystalline lenses: age-related changes and location-dependent variations.</a> Ultrasound Med Biol. 39 (6), 1120-1127 (2013).</li><li>Reilly, M. A., Hamilton, P., Gavin, P., Nathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+behavior+of+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior of natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88 (3), 483-494 (2009).</li><li>Mekonnen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+capsule+significantly+affects+the+viscoelastic+properties+of+the+lens+as+quantified+by+optical+coherence+elastography.">The lens capsule significantly affects the viscoelastic properties of the lens as quantified by optical coherence elastography.</a> Front Bioeng Biotechnol. 11, 1134086 (2023).</li><li>Wilde, G. S., Burd, H. J., Judge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+modulus+data+for+the+human+lens+determined+from+a+spinning+lens+test.">Shear modulus data for the human lens determined from a spinning lens test.</a> Exp Eye Res. 97 (1), 36-48 (2012).</li></ol>

The authors have no conflicts of interest to declare.

Lens compression is a versatile method for estimating lens stiffness. The procedures described above allow comparison between lenses of different species and different sizes. All deformations are normalized against lens size, and the calculation of the elastic modulus approximately accounts for lens size. The effective modulus is considerably higher than the modulus reported previously for the porcine lens4,7,11,19, at least in part due to the use of thickness rather than the radius of curvature: the porcine lens&#39; polar radii of curvature are significantly larger than half the thickness20.
The simple analysis (i.e., use of the Hertz model) presented here has several key limitations. First, it does not account for the presence of the lens capsule. It has been shown that the presence of the capsule may significantly alter the biomechanical properties of the lens16,21. Therefore, this method is best applied to decapsulated lenses. This is particularly important when comparing species in cases where the capsule may have significantly different thicknesses or biomechanical properties. This method also assumes that the lens is mechanically homogeneous; we and others have previously shown that this is generally not the case for porcine or human lenses4,5,6,10,11,22. Thus, it is best to consider the elastic modulus value calculated as an effective modulus, which is presumably related to the volumetric average of the spatially varying modulus within the lens. The Hertz model assumes that the lens is linearly elastic, whereas it is known to be viscoelastic; thus, the simple analysis proposed here is incapable of providing information regarding lens viscoelasticity. Previous work has also shown that the method and duration of lens storage prior to testing can alter lens properties4; all porcine lenses were therefore tested immediately following dissection upon arrival in the lab.
The difference in the force-displacement measurements is due to noise from the load cell amplifier: removal of the capsule makes the force measurements considerably lower, and therefore the signal-to-noise ratio is lower. The assumptions used in deriving the Hertz model importantly include that the sphere is a homogeneous material; therefore, the effective elastic modulus is somehow averaging the deformability of the lens and its capsule when the capsule is present. This makes inter-species and inter-age comparisons particularly difficult because a porcine lens has a capsule ~60 &#181;m thick, whereas a mouse or human lens has a capsule in the 5-15 &#181;m thick range. The elastic modulus of the capsule may also vary with species and age, though these dependencies are unknown. Thus, while it is possible to get a less noisy fit with the capsule, the comparison is inherently confounded by the presence of the capsule-this is the reason we recommend performing the test without the capsule.
Finally, the effective modulus was computed assuming that the radius of curvature was half the thickness of the lens. This is true only for a spherical lens; the porcine lens is significantly aspherical and so the effective modulus values are considerably higher than they would be if the radius of curvature was used instead. This last assumption can be overcome by measuring the radii of curvature, though this can be complicated for the lower surface which is always flat due to contact with the lower plate. It is also less important for more spherical lenses such as murine lenses. Better still is the use of inverse finite element analysis to ascertain the mechanical properties of the lens16.

The lens is the transparent and flexible organ found in the eye that allows it to focus on different distances by changing its refractive power. This ability is known as accommodation. The refractive power is altered due to the contraction and relaxation of the ciliary muscle. When the ciliary muscle contracts, the lens thickens and moves forward, increasing its refractive power1,2. The increase in refractive power allows the lens to focus on nearby objects. As humans age, the lens becomes stiffer and this ability to accommodate is gradually lost; this condition is known as presbyopia. The mechanism of stiffening remains unknown, at least in part due to the difficulties associated with the biomechanical characterization of the lens.
A variety of methods have been employed to estimate lens stiffness and biomechanical properties. These include lens spinning3,4,5, acoustic methods6,7,8, optical methods such as Brillouin microscopy9, indentation10,11, and compression12,13. Compression is the most accessible experimental technique as it can be performed with simple instrumentation (e.g., glass coverslips14,15) or a single motorized stage. We have previously shown how the biomechanical properties of the lens may be rigorously estimated from a compression test16. This process is technically challenging and requires specialized software not readily accessible to lens researchers interested in relative stiffness measurements. Therefore, in the present study, we focus on accessible methods for estimating the elastic modulus of the lens while accounting for lens size. The elastic modulus is an intrinsic material property related to its deformability: a high elastic modulus corresponds to a stiffer material.
The test itself is a parallel plate compression test and can therefore be performed on suitable commercial mechanical testing systems. Here, a custom instrument was constructed comprised of a motor, linear stage, motion controller, load cell, and amplifier. These were controlled using custom software which also recorded time, position, and load at regular intervals. Pig lenses do not accommodate but are easily accessible and inexpensive17. The following method was developed to incrementally compress the eye lens and quantify its elastic modulus. This method can be easily replicated and will be useful in the study of lens stiffness.

<table><tbody><tr><td>Curved Medium Point General Purpose Forceps</td><td>Fisherbrand</td><td>16-100-110</td><td></td></tr><tr><td>Galil COM Libraries</td><td>Galil Motion Control</td><td></td><td></td></tr><tr><td>High Precision Scalpel Handle&amp;nbsp;</td><td>Fisherbrand</td><td>12-000-164</td><td></td></tr><tr><td>Linear Stage</td><td>McMaster-Carr</td><td>6734K4 0.125&quot;</td><td></td></tr><tr><td>Load Cell</td><td>FUTEK</td><td>LSB200-FSH03869</td><td></td></tr><tr><td>Load Cell Amplifier</td><td>FUTEK</td><td>IAA300-FSH03931</td><td></td></tr><tr><td>MATLAB</td><td>The Mathworks, Inc.</td><td></td><td></td></tr><tr><td>Microprobe</td><td>Surgical Design&amp;nbsp;</td><td>22-079-740</td><td></td></tr><tr><td>Miniature Self Opening Precision Scissors&amp;nbsp;</td><td>Excelta&amp;nbsp;</td><td>63042-004</td><td></td></tr><tr><td>Motion Controller</td><td>Galil Motion Control</td><td>DMC-31012</td><td></td></tr><tr><td>Motor</td><td>Galil Motion Control</td><td>BLM-N23-50-1000-B</td><td></td></tr><tr><td>Straight Hemastats&amp;nbsp;</td><td>Fine Science&amp;nbsp;</td><td>NC9247203</td><td>stainless steel, 14cm&amp;nbsp;</td></tr></tbody></table>

automated compression testing of the ocular lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

Six porcine lenses were compressed, first with the capsule intact, then after careful removal of the capsule. Thickness values were 7.65 &#177; 0.43 mm for encapsulated lenses and 6.69 &#177; 0.29 mm for decapsulated lenses (mean &#177; standard deviation). A typical loading history is shown in Figure 3. The resulting force-displacement curves were well-fitted by the Hertz model (i.e., they had a force proportional to the displacement raised to the power of 1.5; Figure 4). This was true for both the encapsulated and decapsulated lenses.
Lenses were first compressed by 15% of their unloaded thickness with an intact capsule, then after removal of the capsule. Axial compression by 15% of the initial thickness has previously been shown not to cause damage to the lens sutures18. Decapsulation resulted in a significant decrease in effective elastic modulus (n = 6; p = 0.0138; Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66040/66040fig01.jpg" /> 
Figure 1: Dissection technique. (A) The extraocular tissues are removed. (B) A circumferential cut is made at the limbus. (C) A circumferential cut is made at the equator. (D) The cornea is removed. (E) The iris is removed. (F) The eye is bisected at the equator, then (G) the vitreous is removed, leaving (H) an annular ring containing the lens, ciliary body, and zonules still attached to the sclera. (I) A meridional cut is made through the sclera to (J) give access to the zonules, (K) which are cut away leaving (L) the encapsulated lens. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/66040fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66040/66040fig02.jpg" /> 
Figure 2: Compression testing apparatus. (A) Schematic and (B) photograph of the lens compression apparatus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/66040fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66040/66040fig03.jpg" /> 
Figure 3: Applied loading history for an encapsulated porcine lens. Top: Displacement history. Bottom: Force history. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/66040fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66040/66040fig04.jpg" /> 
Figure 4: Typical force-displacement data fitted with the Hertz model. Left: Data for an encapsulated porcine lens. Right: Compression data for the same lens after decapsulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/66040fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66040/66040fig05.jpg" /> 
Figure 5: Box and whisker plot of encapsulated and decapsulated porcine lens effective elastic moduli. The effective modulus of the encapsulated lenses was significantly higher than those of the decapsulated lenses (p = 0.013), indicating that the presence of the capsule can substantially alter the effective stiffness of the lens. Data are for six lenses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/66040fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: MATLAB application to control the lens compression apparatus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/Supplemental Coding Files 1.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 2: MATLAB function to estimate the elastic modulus from force-compression data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66040/HertzModulus.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Understanding of Age-Related Lens Stiffness Changes at JoVE.com

Author Spotlight: Advancing Understanding of Age-Related Lens Stiffness Changes 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

automated-compression-testing-of-the-ocular-lens

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.2K Views.  The Ohio State University. We proberen te begrijpen hoe mensen hun gezichtsvermogen verliezen naarmate ze ouder worden. We weten dat dit gedeeltelijk te wijten is aan een verandering in de stijfheid van de lens, maar we weten nog steeds niet waarom de lens verstijft naarmate we ouder worden. De huidige experimentele methoden zijn gebaseerd op ruwe methoden voor het laden van de lens, zoals het plaatsen van microscoopafdekglaasjes op de lens.Deze methoden zijn vervelend en missen de juiste controles voor de laads...

Video: Auteur in de schijnwerpers: Voortschrijdend begrip van leeftijdsgebonden veranderingen in lensstijfheid 

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Chemistry

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Medicine

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

Biology

Binocular Dynamic Visual Acuity in Eyeglass-Corrected Myopic Patients

Current clinical visual assessment mainly focuses on static vision. However, static vision may not sufficiently reflect real-life visual function as moving optotypes are frequently observed daily. Dynamic visual acuity (DVA) might reflect real-life situations better, especially when objects are moving at high speeds. Myopia impacts static uncorrected distance visual acuity, conveniently corrected with eyeglasses. However, due to peripheral defocus and prism effects, eyeglass correction might affect DVA. The present research demonstrates a standard method to examine eyeglass-corrected DVA in myopia patients, and aimed to explore the influence of eyeglass correction on DVA. 
Initially, standard subjective refraction was performed to provide the eyeglass prescription to correct the refractive error. Then, binocular distance vision-corrected DVA was examined using the object-moving DVA protocol. Software was designed to display the moving optotypes according to the preset velocity and size on a screen. The optotype was the standard logarithmic visual chart letter E and moves from the middle of the left to the right side horizontally during the test. Moving optotypes with randomized opening direction for each size are displayed. The subjects were required to identify the opening direction of the optotype, and the DVA is defined as the minimum optotype that subjects could recognize, calculated according to the algorithm of logarithmic visual acuity.
Then, the method was applied in 181 young myopic subjects with eyeglass-corrected-to-normal static visual acuity. Dominant eye, cycloplegic subjective refraction (sphere and cylinder), accommodation function (negative and positive relative accommodation, binocular cross-cylinder), and binocular DVA at 40 and 80 degrees per second (dps) were examined. The results showed that with increasing age, DVA first increased and then decreased. When myopia was fully corrected with eyeglasses, a worse binocular DVA was associated with more significant myopic refractive error. There was no correlation between the dominant eye, accommodation function, and binocular DVA.

The present research demonstrates a method to accurately examine dynamic visual acuity (DVA) in myopic subjects with eyeglass correction. Further analysis indicated that the closer the refraction state to emmetropia, the better the eyeglass-corrected binocular DVA is at both 40 and 80 degrees per second.

Current clinical visual assessment mainly focuses on static vision. However, static vision may not sufficiently reflect real-life visual function as ...

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. 
In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. 
The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

The goal of this protocol is to evaluate changes in metabolic activity and refractive function of the lens in response to experimental treatment.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical ...

Automated Compression Testing of the Ocular Lens

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