作者想感谢她的帮助与生产该视频文章吉特沃纳博士。这项研究是由美国国家科学基金会职业奖。

第1部分：实验准备已批准的机构动物护理和使用委员会在波士顿大学鲎进行的实验程序。动物从海洋生物实验室（伍兹霍尔，MA）或其他供应商购买和住在曝气盐水池，接触到一个稳定的明暗周期的一个房间。引气蟹的生物钟，每天骑自行车之间的白天和夜间的状态的眼睛是非常重要的照明方案。任何侵入性的程序开始之前，立即冷冻动物是在10-15分钟的冰桶中，直至其运动速度减慢，然后插入prosoma两个不锈钢螺钉和opisthosoma固定在一个木制的平台。该平台是用花岗岩加权下方，以便它在汇水。终端实验动物后，再浸入冰浆和pithing的大脑，这首先在于用手术刀口，是安乐死。 第2部分：解决方案鲎林格的解决方案都由430毫米氯化钠，9.56 mM的氯化钾，9.52 mM的氯化钙，9.97 mM的氯化镁，21.05 mM的硫酸镁，50μm的工商业污水附加费，50μMHEPES，和10毫升/大号笔，链球菌混合（青霉素，链霉素，10000个单位/毫升） 。 第3部分：电图记录此过程所需的工具，包括螺丝刀，凡士林，林格氏液，移液管，绿色LED，录音室，不锈钢螺丝，和棉签。 <ol><li>一个手工制作室是用来记录视网膜电图（图1A）。室体的目的是在与眼睛接触生理盐水水库举行。盖子包含一个放大器和一个小洞，大小，以容纳一个LED耦合导电溶液，氯化银导线。一个超亮LED峰值发射520nm左右效果最好。 </li><li>启动，室底部用凡士林涂在眼用两个螺钉固定。 </li><li>会议厅内充满生理盐水，并与盖封顶。 </li><li>后室附件螃蟹放置在一个充满海水通过鳃坦克轻紧笼。 </li><li> LED电缆插入室盖。 </li><li>信号线裁剪氯化线，基准导线夹植入的螺钉。 </li><li>两个导线连接到高阻抗差动放大器的头阶段进行信号放大和放大器的过滤器设置为10Hz以下的频率传递（X -细胞3X4，金控公司）。 </li><li>放大器的输出被送入示波器观看，并为计算机分析和存储的数据采集板。 </li><li>安装后的笼子门是封闭块室内光线下，从到达的动物。 </li><li>数据收集与LabVIEW中编写的自定义程序。该方案的LED闪烁，记录诱发的视网膜电图信号，措施的峰 - 峰值响应振幅。闪光范式的选择取决于实验的目标。例如，一个简短的（100毫秒）的脉冲5V应用到LED，每10分钟监测昼夜变化在眼的灵敏度时，避免了光适应的影响。 </li></ol>第4部分：视神经记录此过程所需的工具，包括螺丝刀，线，环钻，林格氏液，rongeurs，vannas剪刀，录音室，弯钳，细针穿刺探头，一个沉闷的手术刀，手术剪，吸电极。 <ol><li>一个手工制作室是用来记录视神经反应（图1B）。香港总商会的内部是直径为2cm -开得好，有一个在底部壁薄的半圆形开槽开放，以适应神经和周围组织隔离。 </li><li>室附件前一个16号针头插入到心脏之间的铰链的肌肉和血〜20CC是从动物排出。放血是没有必要的，但容易使视神经清扫。 </li><li>视神经的位置估计图纸上的甲壳略呈弧形线之间的横向和中位数的眼睛。 </li><li>一个圆孔，然后在甲壳用环钻切。孔室以及相同的直径。孔的中心位于2-3厘米，前侧眼，稍背线，使神经沿井的腹侧部分运行。 </li><li>直到神经是完全可见，从周边及相关组织，结缔组织清除。 </li><li>周围神经循环的线程链，并进入会议厅拉通过底部的插槽。通过这个开放的神经腔拉弦上轻轻地引导。室以及同时插入孔。然后贴在会议厅内的甲壳用螺丝以及充满林格氏液。 </li><li>室附件后的动物被放置在一个充满海水通过鳃坦克轻紧笼。 </li><li>该井是可视化的下一个立体（SMZ - 168，杰德佩拉公司）和棉花是围绕在底部的开放软垫，以防止血液渗漏和生理盐水出室。 </li><li>罚款的剪刀和镊子残留周围神经的结缔组织被删除，并与新鲜林格氏液加气室。 </li><li>一个尼克是在封装的神经血管，并通过这种开放的神经仔细沿其长度用细剪刀，镊子，针探头desheathed。 </li><li>一个微小的纤维束是从最远的从眼睛传入纤维录制和传出神经纤维录制的眼睛最接近最终在年底使用的探头和切断的神经分离。视频中的传入纤维录音被切断。 </li><li>充满林格解决方案的一个microsuction电极（AM系统公司）是用于视神经记录。电极尖端装有神经束抛光一个直径为1mm高硼硅玻璃毛细管火。 </li><li>电极尖端降低到手动操纵室（WPI的公司），并吸入提示切割的纤维束。提供一个Gilmont注射器连接管电极吸。 </li><li>一个BNC连接提供了信号线和一个氯化银电极周围包裹的导线，以降低噪声提供了参考导致。两根导线连接到一个差分放大器进行信号放大和噪音过滤（X -细胞3X4，金控公司）的头阶段。放大器的输出被发送到示波器查看和计算机分析和存储的数据采集卡。 </li><li>数据收集与LabVIEW中编写的自定义程序。程序控制光刺激，通过数字视频处理器（位+ +剑桥研究系统公司）和数字穗鉴别“（APM，金控公司）记录穗列车接口。通过光纤导光管或整个眼，光可交付单细胞通过电脑控制的视频显示在眼。 </li></ol>第5部分：视网膜内的录音此过程所需的工具包括：一把螺丝刀，一个L形有机玻璃的平台，带螺纹的螺丝孔，用玻璃吸管电极持有人，不锈钢螺丝，镊子，并罚款手术刀。 <ol><li>一个手工制作的有机玻璃板预设的螺丝孔是用来记录细胞内的反应。孔间距安装一个电动显微（PPM5000，WPI的公司）。该板块是动物用两个螺丝固定在侧面和顶部。 </li><li>板附件后的动物被放置在一个充满海水通过鳃坦克轻紧笼。 </li><li>微定位器是拧入板，其动产通过眼睛对准手臂。 </li><li>一批玻璃微量拉，从1毫米外径硼硅玻璃，放置在3M KCl溶液的小瓶微量的秘诀是通过毛细作用回填。 </li><li>其余的移液器，然后手动填写的盐溶液，插入电极支架。 </li><li>电极支架连接到细胞内放大器的头贴微定位臂的阶段（IR - 283，天鹅科技有限公司）。 </li><li>一个微小的视网膜节（〜1平方毫米），用刀片切割角膜接口暴露。 </li><li>林格氏液下降暴露视网膜上，以防止干燥，并通过开放的先进的微管尖端进入视网膜组织。 </li><li>当针尖进入细胞内放大器的电流注入模式的解决方案，是从事和测量电极的阻抗。阻抗20-70兆瓦的范围之外的微量移液器被丢弃。在此范围内的先进的微米步骤，并进入细胞通过电子或机械振动枪头刺穿。 </li><li>三种类型的细胞，可能会遇到的眼睛。 Retinular细胞只有去极化的反应要轻，偏心细胞显示坐上去极化反应的行动潜力的火车，和色素细胞，表明在没有光的反应。 </li><li>数据收集与LabVIEW中编写的自定义程序。该方案通过数字视频处理器（位+，剑桥研究系统）控制的光刺激。刺激传递到光纤管道或视频显示刺穿细胞。在示波器上观察电压响应和数字化计算机程序。 </li></ol>第6部分：代表结果有代表性的视网膜电图如图2a所示。波形代表总结电气主要photoreceptive retinular细胞，因为它们的数量大大超过偏心细胞和电耦合他们。有没有从多个不同的视网膜细胞类型与哺乳动物视网膜电图波形成分。从鲎脑的时钟昼夜反馈调节每天的基础上的视网膜细胞的生理特性，造成的ERG的幅度和时间的过程中，随时间（1）。如图2b所示，ERG振幅是在动物的主观晚上和最低最高在主观上一天。 图3显示了一个代表一个单一的视神经纤维反应轻跟踪。偏心细胞都表现大致相同，呈现出瞬态尖峰放电率增加，通过持续的衰变。率衰减反映（2）偏心细胞的光适应和穗依赖自我抑制的联合行动。秒杀其他的模式，如光依赖率跌幅，被视为在鲎的大脑，而不是眼睛（3）。 代表一个细胞色素细胞，retinular细胞，和偏心光电压响应痕迹如图4所示。只有后两个视网膜细胞类型的视觉享受。的幅度和时间的过程中，他们的反应取决于录音的质量和细胞内的电极的位置。通常情况下，电极穿透色素细胞或retinular细胞，因为其庞大的规模和数量。如果是后者，是一个短暂的去极化衰变持续录得。衰减是由于retinular细胞的光适应（2）。如果电极进入一个古怪的细胞，大动作电位记录在轴突（40 - 70mV）和小的动作电位，坐上去极化的波形附近的SOMA（&lt;25mV的）。 <img src="/files/ftp_upload/1384/1384fig1.jpg" alt="图1" /> 图1。电图记录（A）和视神经记录（二）用于商会的示意图。酒吧等于7毫米在一个和5mm在B <img src="/files/ftp_upload/1384/1384fig2.jpg" alt="图2" /> 图2例跟踪的一个100ms引起了鲎ERG的LED 5V的脉冲在黑暗中（A）和ERG振幅峰-峰值比在不断的黑暗时间波动（二）。黑暗是白色三角形表示的时间开始。 ERG振幅通过由黑色三角形表示的时间的增长，是由于对光线的适应，从而增加了在黑暗中眼的灵敏度。此后ERG振幅的周期性变化是由于动物的内部生物钟。在B时间点，每5min。 <img src="/files/ftp_upload/1384/1384fig3.jpg" alt="图3" /> 图3。视神经纤维反应的跟踪光芒闪过一个单一的ommatidial受体。上述跟踪波形显示刺激的时机。偏心细胞，引起神经纤维的轴突，所有发射实验（5秒闪光灯在完全黑暗的）的光照条件下，因为这显示一个类似的模式。尖峰视网膜编码与哺乳动物共同的属性鲎，不像其他无脊椎动物。 <img src="/files/ftp_upload/1384/1384fig4.jpg" alt="图4" /> 图4电压响应示例痕迹三种类型的细胞在鲎侧眼：光色素细胞（A）retinular细胞（B），和偏心单元（C）。非可视化的第一个单元格。后两个去极化后，一个灯闪。去极化是最大retinular细胞的，因为他们转导的光，通过偏心细胞间隙连接的信号，一路上与一些损失。在偏心细胞，轴突发射动作电位看到骑在穗反向传播到SOMA由于去极化。为更好地观看去极化电位，动作电位的幅度已衰减的数字。细胞的静息电位为- 50mV的。

<ol>
	<li>Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+the+Limulus+visual+system.">Circadian rhythms in the Limulus visual system.</a> J. Neurosci. 3, 856-870 (1983).</li><li>Passaglia, C. L., Dodge, F. A., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+based+model+of+the+Limulus+lateral+eye.">Cell based model of the Limulus lateral eye.</a> J. Neurophysiol. 80, 1800-1815 (1998).</li><li>Snodderly, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+visual+inputs+by+the+brain+of+Limulus.">Processing of visual inputs by the brain of Limulus.</a> J. Neurophysiol. 34, 588-611 (1971).</li><li>Passaglia, C., Dodge, F., Herzog, E., Jackson, S., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+a+neural+code+for+vision.">Deciphering a neural code for vision.</a> Proc. Natl. Acad. Sci. 94, 12649-12654 (1997).</li><li>Barlow, R. B., Kaplan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limulus+lateral+eye:+properties+of+receptor+units+in+the+unexcised+eye.">Limulus lateral eye: properties of receptor units in the unexcised eye.</a> Science. 174, 1027-1029 (1971).</li><li>Bolbecker, A. R., Lewis, A. R., Swan, A. A., Carlson, K., Fleet, J. R., Beck, K. E., Wasserman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Bellows+Cup+Electrode+Demonstrates+Low-frequency+Properties+of+Long-term+Electroretinographic+Recordings+in+the+Limulus+Lateral+Eye.">Stable Bellows Cup Electrode Demonstrates Low-frequency Properties of Long-term Electroretinographic Recordings in the Limulus Lateral Eye.</a> J. Neurosci. Meth. 159, 252-260 (2007).</li></ol>

我们已经演示了如何执行ERG的录音，视神经录音，并在体内的马蹄蟹的视网膜内的录音。每个录音技术都提供了视觉神经基础不同的见解，他们都可以被用来研究在视网膜功能的活的动物感谢螃蟹的大眼睛和坚硬甲壳。视神经活动，甚至可以自由表现在海洋动物电极（4）正确的施工记录。这些技术也可以进行切除眼睛轻微修改设置。自然条件等实验的相关性有限，为的蟹的眼睛变化的生理属性时...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>LED</td>
<td>Light source</td>
<td>Newark Inc</td>
<td>33C1292</td>
<td>&nbsp;</td>
<tr>
<td>Suction electrode</td>
<td>Electrode</td>
<td>A-M Systems, Inc</td>
<td>573000</td>
<td>&nbsp;</td>
<tr>
<td>XCell 3*4-Channel Extracellular Amplifier</td>
<td>Amplifier</td>
<td>FHC Inc.</td>
<td>40-40-8B</td>
<td>&nbsp;</td>
<tr>
<td>Intracellular Recording</td>
<td>Amplifier</td>
<td>Cygnus</td>
<td>IR-283A</td>
<td>&nbsp;</td>
<tr>
<td>APM</td>
<td>Neural Spike Discriminator</td>
<td>FHC Inc.</td>
<td>APM</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Video Board</td>
<td>Cambridge Research Systems Inc</td>
<td>Bits++</td>
<td>&nbsp;</td>
<tr>
<td>Piezopatch Manipulator</td>
<td>Micropositioner</td>
<td>WPI Inc</td>
<td>PPM5000</td>
<td>&nbsp;</td>
<tr>
<td>Square Pulse Stimulator</td>
<td>Nerve Stimulator</td>
<td>Grass Instrument Division, Astro-Med, Inc.</td>
<td>Model S48</td>
<td>&nbsp;</td>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>Model P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate Glass Capillary</td>
<td>Electrode glass</td>
<td>WPI Inc</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Horseshoe crab (Limulus polyphemus)</td>
<td>Animal</td>
<td>Marine Biological Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette Puller</td>
<td>Glass Puller</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Zoom Stereoscope</td>
<td>Microscope</td>
<td>Jed Pella Inc.</td>
<td>SMZ-168</td>
<td>&nbsp;</td>		</tbody>
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using the horseshoe crab, limulus polyphemus, in vision research

美国鲎， 鲎波吕斐摩斯是地球上最古老的动物之一，与动物继续在生物医学研究中发挥了不可或缺的作用。不仅他们的血液中含有特殊的细胞，科学家们使用我们的药物检测bacteriotoxins，但他们的眼睛还包含一个神经网络，提供了有关在我们的视觉系统的生理过程，如光适应和侧抑制，很大的启示。鲎仍然是一个视觉研究的吸引力模型，因为动物是大型和强壮的无脊椎动物，其视网膜神经细胞又大又方便，其视觉系统是紧凑和广泛的研究，其视觉行为是明确界定。此外，眼睛的结构和功能上由一个动物的大脑中的生物钟，每天调制。总之，马蹄蟹的视觉系统是很简单的理解还够复杂，很有趣。 在这个视频中，我们目前调查的基础上，可在体内进行与鲎视觉神经的电生理模式。他们是电图记录，录音视神经和视网膜内的录音。电图（ERG）记录与衡量一个表面电极的电反应在一道闪光的眼睛的所有细胞中总结。它们可以被用来监测时间的延长期的整体灵敏度的眼睛。视神经录音衡量单一的神经纤维与外microsuction电极的扣球活动。它们可以被用来研究从眼睛传达到大脑以及昼夜时钟消息反馈从大脑到眼睛的视觉信息。视网膜内的录音与细胞内微电极测量电压波动引起的光在眼睛的单个细胞。它们可以被用来阐明视网膜处理的细胞机制。

Watch this Scientific Journal Video about Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research at JoVE.com

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

在这个视频中，我们执行与美国鲎电图记录，录音视神经和视网膜内的录音，<em&gt;鲎波吕斐摩斯</em&gt;。这些电的模式，可用于调查的视野，在研究或教学实验室的神经基础。

马蹄蟹，鲎波吕斐摩斯，在视觉研究

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in ...

using-the-horseshoe-crab-limulus-polyphemus-in-vision-research

Research

JoVE Journal

Biology

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

14.2K 次观看.  Boston University. 在这个视频中，我们执行与美国鲎电图记录，录音视神经和视网膜内的录音，鲎波吕斐摩斯。这些电的模式，可用于调查的视野，在研究或教学实验室的神经基础。

视频: Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

从美国的马蹄蟹，鲎波吕斐摩斯采血车

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

科研

生物学

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

使用机器视觉方法的 TEM 工作流程、结果分析和数据管理

A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate thousands of images with numerous parameters that require time-consuming and complicated analysis. AXON synchronicity is a machine-vision synchronization (MVS) software solution designed to address the pain points inherent to TEM studies. Once installed on the microscope, it enables the continuous synchronization of images and metadata generated by the microscope, detector, and in situ systems during an experiment. This connectivity enables the application of machine-vision algorithms that apply a combination of spatial, beam, and digital corrections to center and track a region of interest within the field of view and provide immediate image stabilization. In addition to the substantial improvement in resolution afforded by such stabilization, metadata synchronization enables the application of computational and image analysis algorithms that calculate&#8239;variables between images. This calculated metadata can be used to analyze trends or identify key areas of interest within a dataset, leading to new insights and the development of more sophisticated machine-vision capabilities in the future. One such module that builds on this calculated metadata is dose calibration and management. The dose module provides state-of-the-art calibration, tracking, and management of both the electron fluence (e-/&#197;2&#183;s-1) and cumulative dose (e-/&#197;2) that is delivered to specific areas of the sample on a pixel-by-pixel basis. This enables a comprehensive overview of the interaction between the electron beam and the sample. Experiment analysis is streamlined through a dedicated analysis software in which datasets consisting of images and corresponding metadata are easily visualized, sorted, filtered, and exported. Combined, these tools facilitate efficient collaborations and experimental analysis, encourage data mining and enhance the microscopy experience.

作者聚焦：用于透射电子显微镜工作流程、结果分析和数据管理的机器视觉方法  

Here, we present a protocol to utilize machine-vision software to stabilize dynamic processes during TEM imaging, while concurrently indexing multiple streams of metadata to each image into a navigable timeline. We demonstrate how this platform enables automated calibration and mapping of the electron dose over the course of an experiment.

透射电子显微镜工作流程、结果分析和数据管理的机器视觉方法

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate ...

Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it&#8217;s important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime.&#160;

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.&#160;&#160;

在研究细胞培养实验室中避免真菌和细菌污染的细胞培养技术和实践

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the ...