The following protocols are accepted under the University of Maryland&#39;s Institutional Animal Care and Use Committee as well as the Institutional Review Board. The protocols follow federal, state and local standards, and the guidelines set out by the University of Maryland Policy on Biosafety.
1. Dissection of Eye Sample
<ol>
	<li>Obtain an eye sample from local slaughterhouse or tissue bank. If an entire eye globe is obtained, immediately extract the lens, attached zonules, and v...

<ol>
	<li>Von 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 Ophthal. 1, 1-74 (1855).</li><li>Schachar, R. A., Black, T. D., Kash, R. L., Cudmore, D. P., Schanzlin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+accommodation+and+presbyopia+in+the+primate.">The mechanism of accommodation and presbyopia in the primate.</a> Ann Ophthalmol. 27, 58-67 (1995).</li><li>Glasser, A., Campbell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Reilly, M. A., Hamilton, P. D., Perry, G., 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=Comparison+of+the+behavior+and+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior and natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88, 483-494 (2009).</li><li>Langenbucher, A., Huber, S., Nguyen, N. X., Seitz, B., Gusek-Schneider, G. C., K&#252;chle, M. <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+accommodation+after+implantation+of+an+accommodating+posterior+chamber+intraocular+lens.">Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.</a> J Cataract Refract Surg. 29 (4), 677-685 (2003).</li><li>Ehrmann, K., Ho, A., Parel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+analysis+of+the+accommodative+apparatus+in+primates.">Biomechanical analysis of the accommodative apparatus in primates.</a> Clin Exp Optom. 91 (4), 411 (2008).</li><li>Pinilla Cort&#233;s, L., 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926 (2015).</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>Eppig, 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=Biomechanical+eye+model+and+measurement+setup+for+investigating+accommodating+intraocular+lenses.">Biomechanical eye model and measurement setup for investigating accommodating intraocular lenses.</a> Z Med Ohys. 23 (2), 144-152 (2013).</li><li>Manns, F., Parel, , 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=Response+of+Human+and+Monkey+Lenses+in+a+Lens+Stretcher.">Response of Human and Monkey Lenses in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 48 (7), 3260 (2007).</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>Besner, S., Scarcelli, G., Pineda, R., 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+Brillouin+Analysis+of+the+Aging+Crystalline+Lens.">In Vivo Brillouin Analysis of the Aging Crystalline Lens.</a> Invest Ophthalmol Vis Sci. 57 (13), 5093 (2016).</li><li>Cortes, L., 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926-2932 (2015).</li><li>Bernal, A., Parel, J. -. M., Manns, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Posterior+Zonular+Fiber+Attachment+on+the+Anterior+Hyaloid+Membrane.">Evidence for Posterior Zonular Fiber Attachment on the Anterior Hyaloid Membrane.</a> Invest Ophthalmol Vis Sci. 47 (11), 4708 (2006).</li><li>Kammel, R., Ackermann, R., Mai, T., Damm, C., Nolte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+Lenses+in+a+Lens+Stretcher.">Pig Lenses in a Lens Stretcher.</a> Optom Vis Sci. 89 (6), 908-915 (2012).</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>D'Antin, J. C., Cortes, L. P., Montenegro, G. A., Barraquer, R. I., Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+portable+manual+stretching+device+to+simulate+accommodation.">Evaluation of a portable manual stretching device to simulate accommodation.</a> Acta Ophthalmol. 93 (255), (2015).</li><li>Pierscionek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Marussich, L., 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+Crystalline+Lens+Volume+During+Accommodation+in+a+Lens+Stretcher.">Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 56 (8), 4239 (2015).</li><li>Martinez-Enriquez, E., P&#233;rez-Merino, P., Velasco-Ocana, M., Marcos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OCT-based+full+crystalline+lens+shape+change+during+accommodation+in+vivo.">OCT-based full crystalline lens shape change during accommodation in vivo.</a> Biomed Opt Exp. 8 (2), 918-933 (2017).</li></ol>

We have devised a novel method to provide an accurate and efficient way of studying the accommodation ability of the lens by utilizing a dual-piece clamping mechanism to couple the stretcher to the sample. During accommodation, the lens relaxes, and the diameter decreases in response to relaxation of the zonular fibers1,2,4,19. The method focuses on this phenomenon by clamping and controlling t...

Accommodation is the process by which the human eye is able to dynamically adjust the shape of its crystalline lens to see objects at far or close distances in sharp focus. Accommodation is an intrinsically biomechanical process. Upon neural stimulus, the ciliary muscles produce a force onto the ciliary body and to the zonule fibers that attach to the circumference of the lens capsule1,2. While there are different theories behind the biomechanics of accommodation, the most widely accepted is the Helmholtz hypothesis. According to the hypothesis, the lens is in a natural stretched state, corresponding to the thinnest shape of the lens which is optimal for the focus of distant objects. To change focus to closer objects, the ciliary muscles contract and the zonular fibers are relaxed. In turn, the lens thickens, increasing the anterior and posterior surface curvatures. This corresponds to an increase in dioptric power which is necessary for near vision, therefore, a shorter focal length1.
The ability to accommodate is compromised over time via a condition named presbyopia. Affecting everyone by age 50, presbyopia makes the eye unable to dynamically change focus from far to close distances3. To combat presbyopia, current methods are passive including corrective lenses and bifocals. While increasing one&#39;s ability to focus on close objects at few planes, such passive treatments cannot restore the dynamic focus ability of the lens4,5. In order to treat presbyopia efficiently, or possibly prevent it, there is an ongoing need to better understand accommodation.
To study lens accommodation, a number of devices have been developed to simulate the phenomenon ex vivo4,6,7,8,9. Spinning disks were first introduced to monitor the stretching of the lens via centrifugal forces8. To more faithfully replicate the phenomenon, lens stretching devices were gradually introduced and innovated. Using a lens stretcher, Manns et al. characterized the force required to accommodate the lens while correlating such to lens power and equatorial diameter9. Current understanding is that the lens stiffens with age, resulting in a reduced change in lens shape in response to an equal force from the ciliary body3,10,11,12.
Current lens stretchers often involve a complex setup, implementing electronics and programmable stretching rates, and requires the entire cadaver eyeball6,7,10,13. This requirement increases the cost per experiment to over $500.00 per eye and decreases sample availability. Here we present a method to replicate lens accommodation at low cost as the eye posterior totals around $200.00. While less sophisticated than many devices used today, the technique is much more cost effective and adoptable without compromising results. This method is centered around a manual lens stretcher (MLS) depicted in Figure 1, and uses a unique clamping system on the zonular fibers and a radial twisting method to expand the diameter of the lens. The physiological accuracy of the protocol is validated by the findings of Bernal et al., who studied the pathway by which the anterior and posterior zonular fibers are connected to the lens capsule14. Using the design of custom shoes which only require the lens, zonule, and ciliary body, we aimed to study lens biomechanics by replicating physiological accommodation.

<table border="0" cellpadding="0" cellspacing="0" style="width:856px;" width="856">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Manual Lens Stretcher</td>
			<td style="width:233px;">Bioniko</td>
			<td style="width:132px;">MLS</td>
			<td style="width:300px;">Different animal species will require different shoe sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Porcine Eye Samples</td>
			<td>George G. Ruppersberger; slaughterhouse</td>
			<td>N/A</td>
			<td>Whole eyeballs were obtained</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Eye Samples</td>
			<td>The National Disease Research Interchange</td>
			<td>N/A</td>
			<td>Posterior poles without corneas were ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors (5 1/2&#39;&#39; Straight)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Forceps (4 1/2&#39;&#39;)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iPhone 6s</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Any imaging system with ~0.1 mm resolution will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hypochorite</td>
			<td>Clorox</td>
			<td>Clorox Regular-Bleach</td>
			<td>Any disinfectant will work</td>
		</tr>
	</tbody>
</table>

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.

Porcine eyes, a common sample for studying presbyopia via lens stretching4,15, were obtained, (n = 10) from a local slaughterhouse and this protocol was used to observe the accommodation ability of the lenses. Figure 5A shows the comparison of the porcine lens before and after stretching via the MLS. There was an average 0.19 &#177; 0.07 mm increase in lens radius when stretched (...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

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 ...

The overall goal of this technique is to provide a more feasible and inexpensive method for accurately studying lens biomechanics. This method can help answer key questions in the ocular biomechanics field about how the lens changes with the loss of accommodation and the onset of presbyopia. The main advantage of this technique is that it accurately monitors the ability of the lens to accommodate at a low material cost and a high procedural robustness.After obtaining the eye sample, firmly grasp the side of the eye and use a razor blade to make a small incision along the side three millimeters away from the cornea deep enough to reach the vitreous. Using sterilized scissors, carefully continue the incision around the circumstance of the eye using the forceps to remove the posterior eye tissue once it has been excised. Isolate the lens, zonules, and the attached vitreous using forceps as necessary to disconnect the zonules and the ciliary body from the ciliary muscles while maintaining connection with the lens.Then remove the excess vitreous so the lens can lay flat on the manual lens stretcher. Take care to keep the lens, zonules, and a minimal amount of vitreous intact while removing the excess vitreous. To assemble the stretcher, insert 10 millimeter shoe bottoms and their corresponding shoe tops into the bottom plate of the stretcher such that a five milliliter gap remains between the back wall of the shoe indent and the shoe itself.Using curved forceps, place the extracted lens face up in the middle of the bottom plate so that the shoes are supporting the lens over the central hole. Snap the corresponding top of the shoes into place clipping only the zonules and the vitreous. Then insert the plates into the plate case and the stopper screw into the hole located on the side of the bottom plate and insert the plate case into the base.To measure the lens, place an imaging system directly above the apparatus at the appropriate distance for obtaining images and place a ruler in the field of view for the accurate sizing and scaling of the images in post processing. Next, firmly yet smoothly rotate the wrench in a clockwise direction to stretch the lens and obtain images of the lens. To ensure sample-to-sample consistency, it is essential to rotate the wrench smoothly while stretching the lens.When all of the images have been acquired, rotate the wrench in the counterclockwise direction to restore the sample to its resting state and obtain the images of the final resting state of the lens. To analyze the data, upload the images to the appropriate image analysis software and use the point to select at least 40 points around the circumference of the lens. Under the analyze menu, select measure to yield the location of each selected point and fit the location points with MathLab to yield a radius and chi square of the fit.Then use the photographed ruler to convert the pixel radius and error into metrics and perform paired two-tailed t-test analysis to compare an individual lens before and after stretching. In this representative analysis of 10 porcine eye samples, the lenses demonstrated an over 4%increase from their original radii validating the lens stretching protocol with both literature consistency and accuracy. When human lenses were tested, a dramatic decrease in the elasticity of the lens was observed in correlation with the age of the donor as expected from previous lens elasticity studies.Once mastered, this lens stretching technique can be completed in less than 10 minutes if it is performed properly. While stretching the lens, it's important to remember to provide a continuous wrenching force until the stopper screw has been reached to minimize the variability between samples. Using this manual lens stretching technique, we can monitor the lens stiffness of patients with a variety of disease pathologies including patients with diabetes, cataracts, or presbyopia.After its development, this technique paved the way for researchers in the field of ocular biomechanics to explore the properties of the lens during the loss of accommodation and/or onset phases of presbyopia. After watching this video, you should have a good understanding of how to use a manual lens stretcher to obtain valuable biomechanical insight about the lens. Don't forget that working with biological tissue can be extremely hazardous and that precautions such as wearing proper laboratory attire and sterilizing all of the instruments prior to use should always be taken while performing this procedure.

simulating-mechanics-lens-accommodation-via-manual-lens

Research

JoVE Journal

Bioengineering

6.7K ビュー.  University of Maryland. The overall goal of this technique is to provide a more feasible and inexpensive method for accurately studying lens biomechanics. This method can help answer key questions in the ocular biomechanics field about how the lens changes with the loss of accommodation and the onset of presbyopia. The main advantage of this technique is that it accurately monitors the ability of the lens to accommodate at a low material cost and a high procedural robustness.After obtaining the eye sample, fi...

ビデオ: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

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...

automated compression testing of the ocular lens

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...

Biomechanical Characterization of Porcine Ocular Lens with Automated Compression

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eye lens dissection: a technique to observe zebrafish lens morphology

This video describes the protocol which is developed to dissect adult and larval zebrafish lenses to analyze cortical lens morphology and structural integrity of the lens...

Eye Lens Dissection: A Technique to Observe Zebrafish Lens Morphology

Automated Compression Testing of the Ocular Lens

automated compression testing of porcine ocular lens and estimation of the lens modulus

Automated Compression Testing of Porcine Ocular Lens and Estimation of the Lens Modulus  

microinjection of recombinant rcas(a) retrovirus into embryonic chicken lens

Author Spotlight: Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens...

Studying Protein Expression During Chicken Lens Development

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optimizing mouse primary lens epithelial cell culture: a comprehensive guide to trypsinization

Author Spotlight: Unlocking the Secrets of Cataracts &amp;#8211; Investigating Redox Repair Enzymes in Lens Epithelial Cells

This manuscript outlines a detailed video protocol for culturing primary lens epithelial cells (LECs), aiming to improve reproducibility and aid research in cataracts and posterior capsule opacification (PCO). It offers step-by-step instructions on lens dissection, LECs isolation, and validation, serving as a valuable guide, especially for newcomers in the...

Cultivation and Characterization of Lens Epithelial Cells

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synthesis of soft polysiloxane-urea elastomers for intraocular lens application

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

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...

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

isolating mouse lens epithelial cells to study lens-related disorders

Isolating Mouse Lens Epithelial Cells to Study Lens-Related Disorders

intraocular lens rotation in cataract surgeries

Intraocular Lens Rotation in Cataract Surgeries  

whole mount imaging to visualize and quantify peripheral lens structure, cell morphology, and organization

Author Spotlight: In-Depth Morphometric Examination and Quantification of Native Lens Structure Using Whole Mount Imaging 

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens...

Preparation and Imaging of Murine Ocular Lens Whole Mounts

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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

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...

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

preparation of chicken lens for microinjection of an rcas(a) retrovirus

Preparation of Chicken Lens for Microinjection of an RCAS(A) Retrovirus

assessment of zebrafish lens nucleus localization and sutural integrity

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior...

mouse ocular lens equatorial region whole mount preparation and confocal imaging to understand the lens structural-developmental relationship

Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship  

lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle...

lens transplantation in zebrafish and its application in the analysis of eye mutants

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Lens development involves interactions with other tissues.  Several zebrafish eye mutants are characterized by an abnormally small lens size.  Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

rotating the intraocular lens to prevent posterior capsular opacification in cataract surgeries

Author Spotlight: Enhancing Visual Outcomes in Cataract Surgery: A Novel Technique to Prevent Posterior Capsular Opacification Through IOL Rotation 

The present protocol describes removing residual epithelial cells by rotating the intraocular lens in extracapsular cataract surgery without extra tools for preventing posterior capsular...

Rotating the Intraocular Lens to Prevent Posterior Capsular Opacification in Cataract Surgeries

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Quantification of Oculomotor Responses and Accommodation Through Instrumentation and Analysis Toolboxes

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements can be observed. VisualEyes2020 (VE2020) was developed based on the lack of customizable software-based visual stimulation available for researchers that does not rely on motors or actuators within a traditional haploscope. This new instrument and methodology have been developed for a novel haploscope configuration utilizing both eye tracking and autorefractor systems. Analysis software that enables the synchronized analysis of eye movement and accommodative responses provides vision researchers and clinicians with a reproducible environment and shareable tool. The Vision and Neural Engineering Laboratory&#39;s (VNEL) Eye Movement Analysis Program (VEMAP) was established to process recordings produced by VE2020&#39;s eye trackers, while the Accommodative Movement Analysis Program (AMAP) was created to process the recording outputs from the corresponding autorefractor system. The VNEL studies three primary stimuli: accommodation (blur-driven changes in the convexity of the intraocular lens), vergence (inward, convergent rotation and outward, divergent rotation of the eyes), and saccades (conjugate eye movements). The VEMAP and AMAP utilize similar data flow processes, manual operator interactions, and interventions where necessary; however, these analysis platforms advance the establishment of an objective software suite that minimizes operator reliance. The utility of a graphical interface and its corresponding algorithms allow for a broad range of visual experiments to be conducted with minimal required prior coding experience from its operator(s).

VisualEyes2020 (VE2020) is a custom scripting language that presents, records, and synchronizes visual eye movement stimuli. VE2020 provides stimuli for conjugate eye movements (saccades and smooth pursuit), disconjugate eye movements (vergence), accommodation, and combinations of each. Two analysis programs unify the data processing from the eye tracking and accommodation recording systems.

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements ...

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.

The Mechanics of Poro-Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

In this work, an in vitro reconstitution approach is employed to study the poroelasticity of actomyosin gels under controlled conditions. The dynamics of the actomyosin gel and the embedded solvent are quantified, through which the network poroelasticity is demonstrated. We also discuss the experimental challenges, common pitfalls, and relevance to cell cytoskeleton mechanics.

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This ...

Quantitative Characterization of Bioink Properties for Continuous DLP Printing

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing parameters of digital light processing (DLP). However, the acquisition of working curves wastes materials and requires high formability of materials, which are not suitable for biomaterials. In addition, the reduction of cell activity due to multiple exposures and the failure of structural formation due to repeated positioning are both unavoidable problems in conventional DLP bioprinting. This work introduces a new method of obtaining the working curve and the improvement process of continuous DLP printing technology based on such a working curve. This method of obtaining the working curve is based on the absorbance and photorheological properties of the biomaterials, which do not depend on the formability of the biomaterials. The continuous DLP printing process, obtained from improving the printing process by analyzing the working curve, increases the printing efficiency more than tenfold and greatly improves the activity and functionality of cells, which is beneficial to the development of tissue engineering.

Author Spotlight: Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing  

This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing ...