以下议定书是接受马里兰大学的机构动物保育和使用委员会以及机构审查委员会。这些议定书遵循联邦、州和地方标准, 以及马里兰大学生物安全政策规定的指导方针。
1. 眼部标本解剖
<ol><li>
从当地屠宰场或组织银行获得一份眼科样本。如果整个眼睛地球仪获得, 立即提取晶状体, 附着 zonules, 和玻璃体。 注意: 下面描述的具体细节与猪和人的眼睛有关。<ol>
<li>使用消毒手术剪刀和镊子, 切除和清除周围的巩膜所有多余的组织。</li>
		<li>牢牢地握住眼睛在它的一侧, 并使用剃刀刀片, 使一个小切口沿眼睛一侧3毫米远离角膜。使伤口深到足以达到玻璃内的眼睛。</li>
		<li>使用剪刀, 小心地沿着切口绕着眼睛的圆周切开。避免刺穿镜片。有代表性的图像显示在图 2A中。</li>
		<li>一旦眼睛的外圆周被切开, 用镊子去除眼睛的后部组织。将晶状体、zonules、睫状体和附着的玻璃体与镊子隔离。有代表性的图像显示在图 2B中。</li>
		<li>使用剪刀和镊子, 取出多余的玻璃, 使透镜可以平放在 MLS 上。 注: 在角膜移植的病例中, 角膜按钮用于外科手术, 而其余的地球则可供研究用。然而, 这部分地球仍然可以用于组织准备的镜头担架设置。如果只获得后部, 只执行步骤 1.1. 4–1.1. 5。</li>
	</ol>
</li>
	<li>在15% 漂白剂溶液中消毒所有使用过的设备30分钟。</li>
</ol>2. 手动镜片担架的试用装配
<ol><li>将 10 mm 的鞋底和相应的鞋顶插入到 MLS 的底部板上, 这样在鞋的缩进和鞋的后壁之间仍然有5毫米的空隙。</li>
	<li>将顶部和底部板对齐, 将板材合在一起;设备现在处于无拉伸位置。</li>
	<li>将板材插入板壳中, 塞入螺钉进入底部板侧的孔中。</li>
	<li>将板壳插入底座, 并将扳手置于所对齐的缩进中。</li>
	<li>顺时针扭扳手, 直到它到达停止螺丝, 以收缩鞋, 并扭转回逆时针返回原来的无拉伸位置的鞋。</li>
</ol>3. 镜头的安装
<ol><li>将10毫米的鞋底插入到 MLS 的底部板中, 使5毫米的间隙保持在鞋缩进和鞋本身的后壁上。</li>
	<li>使用弯钳, 将所提取的透镜面向上放置在底部板的中间, 使鞋在中心孔上支撑透镜。</li>
	<li>把相应的鞋顶放到位, 只修剪 zonules 和玻璃。视觉上确保镜头保持在底部板尽可能居中。</li>
	<li>重复步骤2.3–2.4。</li>
</ol>4. 镜片的测量
<ol><li>将成像系统直接放在设备的上方, 以便捕捉视频和拉伸过程的图片。一定要在图片的框架中包括一个标尺, 以便在后处理中准确地调整图像大小和缩放。 注: 任何合适的成像系统都足以进行此步骤;在这里, 我们使用一个12像素, 调焦智能手机一脚从样本。</li>
	<li>牢固而平稳, 旋转扳手在顺时针方向伸展透镜。图 3在无拉伸和拉伸状态下显示具有代表性的图像。</li>
	<li>拍摄拉伸透镜后, 旋转扳手在逆时针方向, 以恢复样品到其静止状态。 注意: 必须及时进行镜片的测量, 以尽量减少镜片的脱水。</li>
	<li>清晰地拍摄镜头的最后静止状态。</li>
</ol>5. 数据分析
<ol><li>将图像上载到 ImageJ, 并使用 "点" 功能在透镜圆周周围至少选择40点, 如图 4A所示。使用 "分析" → "度量" 选项来产生每个选定点的位置。</li>
	<li>适合 (使用软件例如MATLAB) 位置点, 以便产生一个半径和卡方的配合, 如图 4B所示。使用拍照的标尺将像素半径和错误转换为度量值。</li>
	<li>执行配对两个尾 t 测试, 以比较一个单独的镜头之前和之后, 从 MLS 伸展。</li>
</ol>

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AB 公司拥有 Bioniko 咨询有限责任公司的所有权权益。

我们设计了一种新的方法, 以提供一个准确和有效的方法来研究镜头的住宿能力, 利用双件夹紧机构, 以夫妇的担架到样品。在住宿期间, 透镜放松, 并且直径减少为响应悬韧带纤维的放松 1, 2, 4, 19.该方法通过夹紧和控制悬韧带纤维的张力来研究这种现象。因此, 必须对 zonules 内的鞋类进行严格的护理, ?...

住宿是人类的眼睛能够动态调整其晶体透镜形状的过程, 可以看到物体在远距离或接近焦距的尖锐聚焦。住宿是一种内在的生物力学过程。在神经刺激作用下, 睫状肌产生一力到睫状体和 zonule 纤维连接到透镜胶囊的圆周上1,2。虽然有不同的理论背后的生物力学的住宿, 最广泛接受的是亥姆霍兹假说。根据该假说, 透镜处于自然拉伸状态, 与透镜最薄的形状相对应, 它最适合于远距离物体的聚焦。为了将焦点转移到更接近的物体, 睫状肌收缩, 悬韧带纤维松弛。反过来, 透镜变厚, 增加前和后表面曲率。这对应于屈光度功率的增加, 这是近视力所必需的, 因此, 较短的焦距为1。
通过一个名为 "视时间" 的条件, 可容纳的能力受到影响。在50岁时影响每个人, 老视使眼睛无法动态地改变焦点从远到接近距离3。为了对抗视镜, 目前的方法是被动的, 包括矫正镜片和双光眼镜。虽然在少数几架飞机上提高了对接近对象的专注能力, 但这种被动处理无法恢复透镜4、5的动态焦点能力。为了有效地治疗大视地, 或可能阻止它, 目前需要更好地了解住宿。
为了研究透镜的住宿, 已经开发了许多设备来模拟这种现象 "体内"4,6,7,8,9。首先引入旋转圆盘, 通过离心力监测透镜的拉伸情况8。为了更忠实地再现这种现象, 镜头拉伸装置逐渐被引入和创新。使用透镜担架, Manns et al。特征是在与透镜功率和赤道直径9相关的情况下容纳透镜所需的力。目前的理解是, 透镜僵硬的年龄, 导致透镜形状的变化, 以响应从睫状体的同等力量3,10,11,12。
目前的镜头担架往往涉及复杂的设置, 实施电子和可编程伸展率, 并要求整个尸体眼球6,7,10,13。这一要求增加每项实验的成本超过每只眼睛500.00 美元, 并减少样品的可用性。在这里, 我们提出了一种以低成本复制镜头住宿的方法, 因为眼睛的后部总计约为200.00 美元。虽然比今天使用的许多设备都要复杂得多, 但这项技术在不影响结果的情况下更具成本效益和收养。此方法以图 1中描述的手动透镜担架 (MLS) 为中心, 并在悬韧带光纤上使用独特的夹紧系统, 并采用径向扭转方法来扩展透镜的直径。贝尔纳et al的研究结果验证了该协议的生理准确性, 研究了前、后悬韧带纤维连接到透镜胶囊14的途径.采用自定义鞋的设计, 只需要透镜、zonule 和睫状体, 我们的目的是通过复制生理调节来研究透镜的生物力学。

<table><tbody><tr><td>Manual Lens Stretcher</td><td>Bioniko</td><td>MLS</td><td>Different animal species will require different shoe sizes</td></tr><tr><td>Porcine Eye Samples</td><td>George G. Ruppersberger; slaughterhouse</td><td>N/A</td><td>Whole eyeballs were obtained</td></tr><tr><td>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><td>Dissecting Scissors (5 1/2&#039;&#039; Straight)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>Tissue Forceps (4 1/2&#039;&#039;)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>iPhone 6s</td><td>Apple</td><td>N/A</td><td>Any imaging system with ~0.1 mm resolution will work</td></tr><tr><td>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

本议定书的目标是以成本效益高、实用性强的方式模仿生理调节的生物力学。住宿是通过睫状体收缩和 zonule 纤维松弛而实现的, 这导致了近视力所需的透镜的增厚。在这里, 我们提出了一个新的, 简单的方法, 通过绷紧的 zonules 连接到镜头胶囊通过一个手动镜头担架 (MLS) 的住宿复制。这种方法监测透镜在承受一致力时所达到的径向拉伸, 并允许比较可伸展的镜片, 使其不能伸展。重要的是, 担架夫妇对 zonules 直接, 而不是对巩膜的眼睛, 因此只需要透镜, zonules, 和睫状体, 而不是整个地球样本。与获得整个地球相比, 这种差异可以显著降低获得捐赠尸体镜片的成本约62%。

猪眼, 一个共同的样本, 研究通过透镜拉伸4,15, 获得了, (n = 10) 从一个地方屠宰场和这个协议被用来观察的住宿能力的镜片。图 5A显示通过 MLS 拉伸前后猪晶状体的比较。在拉伸 (p < 0.001) 时, 透镜半径平均增加了 0.19 0.07 毫米, 相当于原来半径的4.2 英寸1.62% 的增加。住宿与透镜弹性相关, 因...

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

用人工晶状体担架模拟透镜体的力学性能

我们提出了一个有效的方法来研究透镜的住宿使用人工晶状体担架。该协议模拟生理调节, 通过拉 zonules 连接在透镜胶囊, 从而, 伸展透镜。

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

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

Research

JoVE Journal

Bioengineering

6.7K 次观看.  University of Maryland. 我们提出了一个有效的方法来研究透镜的住宿使用人工晶状体担架。该协议模拟生理调节, 通过拉 zonules 连接在透镜胶囊, 从而, 伸展透镜。

视频: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

伸展的DNA短序列恒力轴向光镊

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

科研

生物工程

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

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.

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

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

化学

Production of a Strain-Measuring Device with an Improved 3D Printer

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in the analog electrical signal in a traditional strain gauge operation, a new strain measurement method is presented here. It uses a photographic technique to display the strain change by amplifying the change of the pointer displacement of the mechanism. A visual polydimethylsiloxane (PDMS) lens with a focal length of 7.16 mm was added to a smartphone camera to generate a lens group acting as a microscope to capture images. It had an equivalent focal length of 5.74 mm. Acrylonitrile butadiene styrene (ABS) and nylon amplifiers were used to test the influence of different materials on the sensor performance. The production of the amplifiers and PDMS lens is based on improved 3D printing technology. The data obtained were compared with the results from finite element analysis (FEA) to verify their validity. The sensitivity of the ABS amplifier was 36.03 &plusmn; 1.34 &#181;&epsilon;/&#181;m, and the sensitivity of the nylon amplifier was 36.55 &plusmn; 0.53 &#181;&epsilon;/&#181;m.

This work presents a strain measurement sensor consisting of an amplification mechanism and a polydimethylsiloxane microscope manufactured using an improved 3D printer.

使用改进的 3D 打印机生产应变测量设备

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in ...

工程学

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

自动压缩猪晶状体的生物力学表征

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.

作者聚焦：增进对与年龄相关的晶状体僵硬变化的理解 

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

人工晶状体的自动压缩测试

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

用人工晶状体担架模拟透镜体的力学性能

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