这项工作是由澳大利亚研究理事会联动基础设施设备和设施格兰特LEO668421和澳大利亚研究理事会联系项目的批准LP0669471的支持。作者非常感谢金泽研究所的技术和横河电机株式会社合作，在建立KIT的麦格理脑研究实验室。

这些准则描述测量利用脑磁图（MEG）学龄前儿童的认知脑功能的设备和程序。首先，我们讨论的一般准则和携带儿童的认知功能的实验测试时必须考虑的问题。其次，我们描述了开发一个协议，以配合4岁儿童的动机和行为能力的脑磁图数据收集的要求。 1。研究与MEG的幼童的一般注意事项清醒的健康儿童工作时，MEG的研究人员面临着一些独特的挑战。首先，一个鲜明和功能的实验室环境是可能的威胁或恐吓幼童。其次，一个实验性的MEG研究的基本要求，包括限制行动，并积极参加长时间任务创造条件，很多成年人觉得乏味和不舒服。子女就读时，就成了当务之急，以适应孩子们的能力和局限性的实验程序。在本节中，我们描述了在准备参加在数据采集会议的主题涉及的初始步骤。 1.1熟悉/培训会议：对于学前儿童，了解/培训会议定于之前的实际数据采集会话。这次会议让孩子熟悉的环境和自己的步调，研究人员。我们的儿童及家长介绍，研究人员和他们熟悉实验室的环境和套路。通过介绍会议结束时，家长和孩子明白在参与涉及的所有步骤，并了解他们应该期望在数据记录。如果首次访问期间，孩子出现害羞或焦虑，我们有一段时间的玩耍，直到他们感觉更舒服。然后，我们引进的所有步骤进行。 1.2环境的解释 ：为了解释梅格环境对孩子，我们采用一个主题，提供了一个儿童友好的理由，对周围环境。例如，MEG系统是飞船等待空间冒险的孩子，其中可能会出现一些不熟悉的事件。实验室也被装饰墙贴和玩具（图1），它有一个专门的的游戏室。孩子说话时，我们使用的语言，孩​​子们可以很容易理解，如&#39;冻结&#39;，而不是“最大限度地减少运动”，“宇航员头盔”，而不是“标记线圈帽”。 1.3解释的程序，给孩子和家长：我们展示的儿童及家长前儿童的内磁屏蔽室（MSR）所采取的步骤。的首要步骤之一是适合主体的头部第一个5个标记线圈，这些线圈表明主体的内部梅格头盔的头的位置。与孩子的帮助下，我们数字化的一个傀儡的头部形状，然后我们邀请孩子和家长陪MSR /飞船的傀儡。一旦内的MSR，我们将木偶的头部，头盔内看电影。然后，我们问孩子，如果他们想观看电影与傀儡。如果孩子是舒适和轻松的和他们的父母是与我们的协议感到满意，我们邀请他们玩一个游戏内的飞船，下一次他们参观我们的实验室。 1.4工作簿：我们有基于太空探险主题，显示儿童测试会议期间将采取的步骤的工作簿。孩子们接受这些worksbooks带回家，并与他们的父母通过他们准备在下次访问。工作簿的目的是强调测试的重要性的三大要求：一）避免任何内MSR有色金属材料，B）放置标记线圈帽，以确保准确的数据共同登记和C）和其余仍然在数字化和数据采集的重要性。 2。一个用于测量岁儿童学前教育的MEG信号的协议 2.1设备的数据采集和实验任务期间使用 标记线圈第：为了配合主体的结构信息（如MRI或数字化的头部形状），一套五线圈MEG的数据被放置在一个孩子穿的MEG头盔内的泳帽。这些线圈作为参考点，之前和之后的多少来衡量主体的头采集过程中提出的任务相比，他们的立场。 数字转换器：数据采集，前五个标记线圈和头部形状的主体地位是数字化，使用Polhemus Fastrak ®数字化仪（科​​尔切斯特，VT）。数字化仪由一支笔数字化仪，3个接收器，发射机和运动跟踪单位。这个过程的措施，线圈的位置。后来，这些位置是相匹配的MEG获得调整所收购的磁性数据和头部形状的位置信息。 儿童脑磁图系统 ：数据采集是使用整个头部的孩子脑磁图系统（KIT，日本金泽）[2] 。数据是不断收购在1000Hz的采样率，与网上的带通滤波器0.03赫兹和200Hz之间。标记线圈位置获得之前和之后的录音测量头部运动。数据都将被拒绝，如果头部运动大于5mm。 脑磁图系统外设：使用演示的PC控制实验刺激®（神经行为系统，奥尔巴尼，CA） 。提供可视化的投入是一台投影仪，坐在外面的MSR和体现到MSR屏幕。提供听觉刺激，音特美研究模型ER - 30插入耳机配备了婴儿大小的泡沫耳塞（音特美研究公司的Elk Grove Village的，白细胞介素）。孩子是不断闭路摄像头和监控实验者通过麦克风的孩子通信。 2.2数据采集：数据记录会涉及到以下几个阶段： 第一阶段：在此之前孩子的到来 重要的是要尽快到达孩子准备开始实验的所有计算机，软件和设备。作为一般建议，两位经验丰富的研究人员应该存在的，而孩子和家长都在实验室。清除研究员，父母和子女之间的通信实验成功的关键。 第二阶段：孩子到达后 <ol><li>签署同意书，和实验者检查，如果家长有疑问。 </li><li>实验者检查，如果孩子记得第一次会议期间或在小册子介绍的所有步骤，并再次引入任何不记得。强调的是数字化和数据采集期间冻结或减少运动的重要性。这可以证明使用一个傀儡。 </li><li>然后，实验者检查，如果孩子携带任何有色金属材料（服装，首饰闪闪发亮的补丁，里面的口袋被遗忘的玩具，发夹等）。如果他们携带了一件衣服，其中包含有色金属材料，我们提供与MEG的安全服。如果家长想陪孩子在实验过程中，我们保证，他们也解除他们的有色金属材料。 </li></ol> 第三阶段：获得孩子准备执行任务里面的MSR <ol><li>实验者适合帽，拥有5个标记线圈。 </li><li>实验者数字化标记线圈的位置和孩子的头部形状。为了保持在一个固定的位置首脑，我们已与3个接收器安装使用颈箍。重要的是要加强，减少运动或“冻结”定期获得了一个好头形状的想法。孩子可能会奖励贴纸做一个&#39;冻结&#39;做好。 </li><li>孩子被邀请进入MSR。耳机插入和孩子是在仰卧位定位梅格杜瓦头盔。 </li><li>一旦孩子与他们的MEG头盔内元首舒适躺下，标志线圈连接到自己的动力装置，MSR门是关闭的。实验者之一仍然是在整个会议期间的房间（和也父母，如果需要的话）。 </li><li>在安装过程中收集数据块之间的娱乐，电影或卡通可投射到MSR背投影屏幕。 </li></ol> 第四阶段：运行实验 <ol><li>一旦MSR门是关闭的，鱿鱼从事数据采集开始。 </li><li>随着孩子看一部电影，一个coregistration测试前进行，以确保脑磁图信号可以数字化的头部形状配准。这个预测试包括匹配梅格系统所示的位置，在数字化过程中获得的标记线圈的位置。 </li><li>一旦在第一次测试，孩子的头是正确的头盔内的位置显示，研究人员可以开始实验：（一）电影是静音;（二）研究人员询问孩子是否准备好，以“玩游戏”; （c）一旦比赛开始，实验者定期提供反馈的孩子。例如，一个孩子可能会被要求听一系列的句子，重复一个目标词，如“奶酪”，只要他们听到它。这些目标的话，嵌入式赶上三集ALS探头持续注意力的任务，但不包括在最后的分析。 </li><li>任务完成后，另一个标志线圈进行测量监控头的位置和运动。 </li><li>鱿鱼是脱离后，MSR的门被打开，孩子被邀请到游戏室，在那里他们可以选择一个良好的工作为他们的奖金。 </li></ol> 3。代表性的成果在我们实验室的一个具有代表性的研究使用此协议来研究儿童语言发展的逻辑。参加二十六个孩子年龄在3-4岁（平均4岁和5个月）中至少有一个收购会话。这些儿童中有17返回第二个测试会话和5个第三个测试会话。平均头部运动，小于3.7毫米（SD 3.1毫米），以及5mm的阈值内，。发生变动大于5mm的变化范围从5.5毫米到15毫米（平均=8.6毫米，SD = 2.5毫米），4.5％的会议。 <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="图1" /> 图1：MSR和MEG头盔装饰贴纸和毛绒玩具。 <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="图2" /> 图2 4岁女孩在成人大小的MEG（左）。注意她的头顶部填充，以尽量减少运动和头部和头盔之间的距离。右图显示了同一个女孩在孩子的MEG。

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, 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+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

此协议旨在促进健康和清醒的4岁儿童的认知脑功能的测量。我们使用自定义尺寸的MEG的系统设计，以适应岁儿童学前教育的主管。有了这个系统，一个儿童友好的实验室环境，和适应儿童的能力和动机的协议，我们能够在一个年龄范围，其中有目前非常少的数据来衡量认知脑功能。 配套麦格理系统是第一个完整的头的系统自定义大小与学前教育岁以下的儿童使用。主要有三个子女就读定制头盔杜瓦的优势。首先，儿童小号头neuromagnetic信号范围半径较小的传感器配置带入鱿鱼传感器。第二，规模较小的头盔让孩子的头完全插入到杜瓦。完全插入是防止成人杜瓦头盔，因为较小的冠承担儿童的距离（见图2）。这两个因素是使用的MEG记录大脑活动的基础，因为neuromagnetic信号随距离迅速衰减。三，定制的儿童的头部对称定位头盔艾滋病和限制孩子的头部运动杜瓦内的自由。这些功能大大方便了从儿童的脑磁图信号的设置，定位和测量。 自定义大小的孩子脑磁图系统的问世，是认知神经科学的一个重要进展。替代性脑功能成像技术，聘请放射性药物或强磁场可能不适合年纪较小的儿童的日常使用。由于MEG的是一个完全被动的测量技术，有发展组织无法想象的风险。目前还很少的MEG的健康学龄前儿童岁以下儿童的数据，因为传统的成人MEG系统不太适合与儿童使用。儿童脑磁图系统将让我们研究大脑功能的大脑活动的一个戏剧性的结构正在发生的年龄，认知功能正常和病理发展提供了新的见解。

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<td>Whole head child MEG</td>
<td>&nbsp;</td>
<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
<td>Model PQ1064R-N2m</td>
<td>&nbsp;</td>
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<td>Magnetically shielded room</td>
<td>&nbsp;</td>
<td>Fujihara Co. Ltd., (Tokyo)</td>
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<td>Digitiser</td>
<td>&nbsp;</td>
<td>Polhemus (Colchester, VT)</td>
<td>Fastrack</td>
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<td>&nbsp;</td>
<td>Neurobehavioral Systems (Albany, CA)</td>
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measurement of neuromagnetic brain function in pre-school children with custom sized meg

脑磁图是一种技术，检测与皮层的活动[1]相关的磁场。大脑电活动产生电场 - 使用脑电图（EEG） - 和其相应的磁场，可以记录 - 脑磁图检测。 MEG的信号是由专门的传感器被称为超导量子干涉器件（鱿鱼）检测。超导传感器需要冷却的液态氦在-270 ° C。它们都包含在一个vacumm绝缘头盔称为杜瓦，这是充满液体。被放置在固定的位置，在氦冷却剂杜瓦内的头盔，鱿鱼和脑磁图测量主体的头放在头盔杜瓦内。头盔杜瓦的大小必须满足反对约束。显然，它必须足够大，以适应大部分或全部人口将要研究的元首。然而，头盔，还必须足够小，保持最微小的，它们是衡量脑领域范围内的鱿鱼传感器。传统的全头MEG系统的设计，以容纳超过90％的成人头。但是成人的系统并不适合测量脑功能的头半径比成年人更小的数厘米在学前教育chidren。 KIT的麦格理Macquarie大学脑研究实验室使用一个MEG系统自定义大小以适合学龄前儿童元首。这孩子系统有64个一阶轴向梯度计50毫米基线[2]，内磁屏蔽室（MSR）与传统的成人大小的MEG系统中[3,4]。主要有三个子女就读定制头盔杜瓦的优势。首先，儿童的头neuromagnetic信号范围半径较小的传感器配置带入鱿鱼传感器。第二，规模较小的头盔让孩子的头完全插入到杜瓦。完全插入是防止成人杜瓦头盔，因为较小的冠，肩儿童的距离。这两个因素是使用的MEG记录大脑活动的基础，因为neuromagnetic信号随距离迅速衰减。三，定制的儿童的头部对称定位头盔艾滋病和限制孩子的头部运动杜瓦内的自由。当一个协议，将数据收集的要求与儿童的动机和行为能力，这些功能大大方便安装，定位，和脑磁图信号的测量。

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

脑磁图系统大小为幼儿的出现打开了重要的新机会，以研究大脑发育。新的系统，连同一个协议，将儿童的能力，实验要求，可用于研究在三至六岁的健康，清醒的儿童的认知和语言的过程。

在学前儿童Neuromagnetic的脑功能自定义尺寸的MEG测量

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

measurement-neuromagnetic-brain-function-pre-school-children-with

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Research

JoVE Journal

Biology

16.8K 次观看.  Macquarie University. 脑磁图系统大小为幼儿的出现打开了重要的新机会，以研究大脑发育。新的系统，连同一个协议，将儿童的能力，实验要求，可用于研究在三至六岁的健康，清醒的儿童的认知和语言的过程。

视频: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

近红外光谱技术在婴儿，儿童及成人的认知功能探索

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

执行自定义的微RNA基因芯片实验

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...