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 c...

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>

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,<sup ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#34;</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&#160;</td>
			<td>22-079-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature Self Opening Precision Scissors&#160;</td>
			<td>Excelta&#160;</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&#160;</td>
			<td>Fine Science&#160;</td>
			<td>NC9247203</td>
			<td>stainless steel, 14cm&#160;</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.

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

Automated Compression Testing of the Ocular Lens

We're trying to understand how people lose their near vision with age. We know that this is due, in part, to a change in lens stiffness, but we still don't know why the lens stiffens as we age. Current experimental methods rely on crude methods for loading the lens, such as placing microscope cover slips on the lens.These methods are tedious and lack appropriate controls for the rate of loading. We have determined that the lens shape is the primary driver of presbyopia, and we know that the shape is primarily driven by biomechanical parameters. This protocol offers a completely objective, reproducible method for measuring lens stiffness.Automation allows us to control the rate of loading, which is a key parameter when characterizing a viscoelastic material, like the lens. We now have an objective, repeatable protocol for measuring lens biomechanical properties. This will allow us to rigorously characterize how the lens stiffens with age, as well as how specific proteins modify the lens biomechanical properties.

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...

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

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

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