This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).

所有动物的程序均按照建议的指南中照顾和实验动物使用由卫生和批准议定书下国家研究院​​的机构动物护理和使用委员会在斯克里普斯研究所进行。 1.解剖镜<ol><li>根据健康"指南实验动物的护理和使用"的国家机构和认可机构使用动物的协议安乐死的建议只。 </li><li>去核的使用弯钳小鼠眼球。按压围绕与镊子眼组织带来的眼睛从插座中，然后从与镊子插座摘去眼。在解剖盘传送眼睛到新鲜的1X磷酸盐缓冲盐水（PBS）中。 </li><li>切断视神经接近眼球越好。轻轻地，小心地穿过孔插入wher细直镊子进入眼球E中的视神经退出的后路。 </li><li>小心使用剪刀切开成从后角膜边缘的眼球。啮齿动物的透镜占据的眼睛的〜30％。请仔细这些切口，不插入镊子或剪刀太深进入眼内，以免损坏镜头。 </li><li>沿角膜和巩膜在围绕眼球至少一半的方式之间的接合处切割。 </li><li>轻轻地推在角膜上通过在步骤1.4和1.5所取得的开口以除去从眼睛的晶状体。 </li><li>使用精尖镊子直小心删除仍然连在镜头任何大的杂物。目视检查是否有损坏镜头在进行硬度测量之前。 </li></ol> 2.测量刚度<ol><li>从使用分析天平同一框体重至少10盖玻片。查找的平均重量的盖玻片。为了保持一致性，使用盖玻片所有实验的同一个盒子。预湿盖玻片和直角镜在1×PBS中在室温下至少2小时开始实验之前。 </li><li>填测量室（参见图1）与65 - 75毫升1×PBS中。测量室由一个内部的机械车间做出来有机玻璃，并在该室草皮被钻床设置为所需的深度与适当的钻头制成。透镜在1×PBS中保持透明，在室温下机械测试的持续时间。 </li></ol><img alt="图1" src="/files/ftp_upload/53986/53986fig1.jpg" /> 图1:刚度测量室示出了各种不同的深度和形状的草皮层的定做刚度测量室的尺寸的照片。圆形凹坑是200微米或300微米深（黄色箭头）被用于对小鼠透镜的测量结果。草皮是2毫米直径和〜13-从腔边缘14毫米<a href="https://www.jove.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>放置直角镜进入室在从将被用于保持透镜的草皮层的恒定距离。确保镜在实验过程中不动。 </li><li>转移解剖镜头测量室仔细抓住钳或弯钳。 </li><li>取卸载透镜的顶视图图片从直接开销。取小鼠透镜照片在30X放大倍数下通过从解剖显微镜（底部），在左，右两侧光纤光源照明。设置光纤电源到最大光强度的80％。根据需要调整基于环境照明，用户偏好和画质电源输出。 </li><li>取卸载透镜的侧视图的图片，这可以通过里可以看到GHT角度的镜子。如果相机未校准，采取在聚焦镜边缘的图片。镜边缘为5mm长，并且该测量可以在以后被用来确定像素/毫米，并作为图像中的比例尺。 </li><li>镜头放置到草皮，并确认镜头在草皮安全，直就位。取装载前透镜的图象。该镜头应在其前或后极部草皮静养。 </li><li>轻轻地放在1盖玻片上的镜头。等待2分钟，以允许蠕变，并采取装载镜头的另一侧查看图片。 </li><li>继续加入盖玻片如在步骤2.8和服用侧视图图片加入每个盖玻片的如在步骤2.8之后，直到总共10盖玻片被应用。 </li><li>删除所有盖玻片。等待2分钟，并除去所有盖玻片后取透镜（内部和外部的草皮的）的侧视图图片。 </li></ol> 3.晶状体核测量<ol><li>要determ的INE晶状体核大小，移动镜头到一个干净的培养皿中充满了1X PBS。 </li><li>用细镊子直轻轻解封镜头。 </li><li>脱落的皮质纤维细胞通过滚动手套的手指之间的镜头。剩下的晶状体核会感觉像一个硬的大理石。使用此过程来隔离成人镜头细胞核起1个月的年龄。因为分离的核是一个刚体，不能用此描述的方法进行晶状体核的进一步的机械测试。 </li><li>轻轻漂洗在培养皿在1×PBS中的晶状体核。 </li><li>放置晶状体核放回测量室（未在草皮），并采取晶状体核的图像通过直角反射镜。 </li></ol><img alt="图2" src="/files/ftp_upload/53986/53986fig2.jpg" /> 图2:恩小鼠镜头由盖玻片压缩 （一）示意图和（B）的照片perimental设置表示在填充用1×PBS测量室200微米深草皮一个2个月大的小鼠透镜。直角反射镜和安装在解剖显微镜的数字照相机中使用由盖玻片压缩过程中收集透镜的图像。 （C）的通过连续增加盖玻片的数字压缩的2个月大的野生型透镜的矢状次照片用于测量轴向和赤道直径和基于盖玻片压缩测试期间计算轴向和赤道菌株提供的原始数据。透镜的反射有时可以在盖玻片（最清晰可见的1盖玻片图像中的）中看到。当进行测量时，忽略该反射并测量到透镜的顶点。 （D）的2个月大的野生型镜头后的压缩和隔离晶状体核矢状美景照片。该压缩后的镜头和孤立的核正坐在草皮之外。比例尺1毫米。这个数字是从Gokh修改在， 等 公共科学图书馆·之一 ，2012 19。 <a href="https://www.jove.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.图像分析<ol><li>装货前和使用ImageJ或类似软件的每一装载步骤后测量镜头赤道和轴向的直径。测量每个晶状体核的直径。晶状体核是近球形所以在任何方向的测量就足够了19,21。 </li><li>通过增加使用的草皮的深度修正镜头直径轴。在测量室中，草皮层遮蔽了透镜的轴向厚度为200微米（2个月大的小鼠透镜）或300微米（4月龄和8个月大的小鼠镜头）。 </li><li>使用方程，ε=（D - D 0）计算从透镜直径测量的轴向和赤道菌株/天0，其中ε为应变，d为轴向或电子邮件quatorial直径在给定的负载，而d 0是在零负载的相应的轴向或赤道直径。 </li><li>画出轴向和赤道株作为施加的负荷（毫克）的功能。 </li><li>绘制轴，赤道和核直径。计算并通过由赤道直径除以轴径绘制透镜纵横比。 </li><li>计算和使用等式绘制透镜体积，体积= 4/3×π×R e 2 的 ×R A，其中R e为赤道半径和R a是从在步骤2.6拍摄的照片测量的轴向半径。这个公式假定镜头是一个扁球体（椭球）1.22。 </li><li>计算并用公式，体积= 4/3×π×R N 3，其中R N是透镜核的半径为从在步骤3.5拍摄的照片测量绘制核体积。这个公式假定晶状体核我SA球19,21。 </li><li>计算并绘制细胞核部分作为核体积与透镜体积的比值。 </li></ol>

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作者什么都没有透露。

使用这种方法来衡量镜片硬度时，有几个关键因素。 （ - 8.5°8）相对于所述腔室（θ）的底部第一，盖玻片在略微倾斜的角度施加到透镜。这将适用于负载的非常小的组分平展，而不是轴​​向。然而，这种赤道负载可以忽略不计，因为罪θ≈0.1 19。如果此方法适于大透镜，盖玻片到腔室的底部的角将需要被测量以确定赤道负载是否应计入应变计算。其次，它是重要的，以允许透镜到加?...

The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses. 
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness. 
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.

<table><tbody><tr><td>Fine tip straight forceps</td><td>Fine Scientific Tools</td><td>11252-40</td><td></td></tr><tr><td>Microdissection scissors, straight edge</td><td>Fine Scientific Tools</td><td>15000-00</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Scientific Tools</td><td>11272-40</td><td></td></tr><tr><td>Seizing forceps</td><td>Hammacher</td><td>HSC 702-93</td><td>Optional</td></tr><tr><td>Dissection dish</td><td>Fisher Scientific</td><td>12565154</td><td></td></tr><tr><td>60 mm Petri dish</td><td>Fisher Scientific</td><td>0875713A</td><td></td></tr><tr><td>1x phosphate buffered saline (PBS)</td><td>Life Technologies</td><td>14190</td><td></td></tr><tr><td>18 x 18 mm glass coverslips</td><td>Fisher Scientific</td><td>12-542A</td><td></td></tr><tr><td>Measurement chamber with divots to hold lenses</td><td></td><td></td><td>Custom-made (see &lt;strong&gt;Figure 1&lt;/strong&gt;)</td></tr><tr><td>Right-angle mirror</td><td>Edmund Optics</td><td>45-591</td><td></td></tr><tr><td>Light source</td><td>Schott/Fostec</td><td>8375</td><td></td></tr><tr><td>Illuminated dissecting microscope</td><td>Olympus</td><td>SZX-ILLD100</td><td>With SZ-PT phototube</td></tr><tr><td>Digital camera</td><td>Nikon</td><td>Coolpix 990</td><td></td></tr></tbody></table>

sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses

眼透镜是透明的器官，折射和光聚焦，以形成在视网膜上的清晰图像。在人类中，睫状肌收缩变形的透镜，从而增加在透镜"光功率集中于附近的物体，被称为住宿的方法。在镜头的刚度与年龄相关的变化都与老花眼​​，在适应镜头的能力降低，以及由此延伸，需要老花镜。尽管鼠标镜头不适合或开发老花眼，小鼠模型可以为理解镜头病症的宝贵基因工具，并在小鼠中观察到加速老化能够在镜头年龄相关的变化研究。这个协议表明用于确定鼠标透镜刚度，用玻璃盖玻片依次施加压缩负荷增大到透镜的简单，精确和成本效益的方法。有代表性的数据证实，鼠标透镜随着年龄的增长变得更硬，像人类的镜头。这种方法是高度可重复的，并且可以潜在地扩大到从较大的动物机械地测试镜片。

刚度和2-，4-和8个月大的小鼠的镜片的尺寸进行了测量。小鼠从TSRI动物育种设施获得的纯C57BL6菌株背景所有野生型动物，并且每一个透镜装载有1至10个盖玻片。轴向和赤道株计算为通过测量透镜的轴向和赤道直径在加入每个盖玻片的后，再归一直径为相应的卸载每个直径变化的施加载荷的功能。从每个年龄八个透镜进行了测试，结果表示为平均值±标准误差。如图先前...

Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

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

盖玻片序贯应用评估鼠标透镜的抗压刚度:应变和形态分析

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 ...

sequential-application-glass-coverslips-to-assess-compressive

Research

JoVE Journal

Biology

7.2K 次观看.  The Scripps Research Institute. 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.

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

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

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens 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.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

科研

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

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.

用人工晶状体担架模拟透镜体的力学性能

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

生物工程

晶状体纤维细胞中膜叉指的免疫荧光染色

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear image. The bulk of this tissue comprises specialized, differentiated fiber cells that have a hexagonal cross section and extend from the anterior to the posterior poles of the lens. These long and skinny cells are tightly opposed to neighboring cells and have complex interdigitations along the length of the cell. The specialized interlocking structures are required for normal biomechanical properties of the lens and have been extensively described using electron microscopy techniques. This protocol demonstrates the first method to preserve and immunostain singular as well as bundles of mouse lens fiber cells to allow the detailed localization of proteins within these complexly shaped cells. The representative data show staining of the peripheral, differentiating, mature, and nuclear fiber cells across all regions of the lens. This method can potentially be used on fiber cells isolated from lenses of other species.

作者聚焦:通过对复杂叉指和蛋白质染色进行成像的新型方案推进晶状体生物力学研究 

This protocol describes methods to prepare peripheral, mature, and nuclear eye lens fiber cells for immunofluorescence staining to study complex cell-to-cell interdigitations and the membrane architecture.

晶状体皮层和晶状体核束和单纤维细胞的制备和免疫荧光染色

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear ...

发育生物学

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 ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

盖玻片序贯应用评估鼠标透镜的抗压刚度:应变和形态分析

摘要

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此视频中的章节

在斑马鱼和镜头移植在眼突变分析中的应用

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

用人工晶状体担架模拟透镜体的力学性能

晶状体皮层和晶状体核束和单纤维细胞的制备和免疫荧光染色

晶状体皮层和晶状体核束和单纤维细胞的制备和免疫荧光染色

晶状体皮层和晶状体核束和单纤维细胞的制备和免疫荧光染色

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进