美国国立卫生研究院NIBIB RO1:1R01EB009103 - 01 临床Neuroengineering培训计划（1 T90 DK070079 - 01） 华莱士H库尔特基金会 美国国立卫生研究院的机构临床和转化科学奖 美国国立卫生研究院/ NCRR 1KL2RR025012 - 01 威斯康星校友研究基金会

实验过程<ol><li>脑电图电极的连接<ol><li>电极会附着在头皮上使用脑电图帽;这简化了确保电极在头皮上的适当位置的过程中，10-20国际体系中指定。 </li><li>若要帽，标志主体的头皮上的顶点使用毡尖笔或一些其他类似的方法。要做到这一点，开始寻找关于这个问题的鼻根和枕外隆凸尖。用一个卷尺，找到这两个地点之间的距离。点之间的两分，或50％的距离，中间是顶点。请，以供日后参考点的标记。其他10-20点可设在类似的方式。 </li><li>查找锆电极帽，和顶点的位置。保持锆石固定，防滑盖到头上。确保FZ，CZ，PZ电极上的头皮中线，O1 - O2电极与OZ的水平和层次，以及FP1，FP2，FPZ的水平。 </li><li>现在，参比电极连接，通常到一个耳垂的剪辑。 </li><li>接下来，电极用导电胶填充，使电极与头皮的低阻抗的接触。要做到这一点，用钝头的针的小注射器充满凝胶。此外，它可在计算机屏幕上看到的脑电图的痕迹，而凝胶被应用，以确定是否连接好。 </li><li>插入电极针，轻轻擦伤了头皮针，以消除任何死皮。填充少量的凝胶电极，小心​​不要过满。开始用耳参比电极，并重复所有电极，包括地面。 </li><li>检查所有通道，都应该小于5kΩ的阻抗。这种方法会有所不同，根据正在使用的特定放大器系统，但它应该有可能通过硬件或通过BCI2000阻抗检查。 </li><li>如果超过5kΩ的电极，再插入针头，和擦伤头皮。它不应该有必要投入更多的凝胶，除非有阻抗没有下降。 </li></ol></li><li>获取脑电图校准功能<ol><li>在会话的开始，尤其是第一次的主题是正在测试，它是有用的，确定可以用于控制一个BCI的脑电图功能。这是因为虽然毛乌素和β脑电节律的基本属性是所有的人一样，这些功能会有所不同从人到人，并因此需要进行校准之前，任何其他的实验。 </li><li>在校准会议，主题是指示想象中不同的动作反应在显示器上呈现的视觉线索，涉及他们的手或脚。要开始，计算机系统应配置双显示器模式，使研究者的显示屏包含了控制软件，并在第二台监视器显示实验输出。 </li><li>从BCI2000Launcher开始选择你的功放源模块，ARSignalProcessing模块和StimulusPresentation模块BCI2000。在这个例子中，我们使用gUSBampSource模块，控制g.USBamp放大器。 </li><li>添加的参数为你的主题文件，放大器和电机的筛查任务。应提前配置这些，使他们可以简单地加载，并可以开始实验。 </li><li>一旦参数文件已被添加到文件列表中BCI2000Launcher中时，按下启动按钮。如果一切运行正常，BCI2000应该启动，应该出现一丝的脑电图数据，和主体的监测实验开始之前应该是空白的。 </li><li>会议期间，屏幕上会是空白的，或显示指令，如"右手"，"左手"，"两手"，或指令3S屏幕上会出现"两只脚。";在这个时候，主体应不断想象的运动。手的动作应该是打开和关闭的手（例如，像挤压网球），和脚的运动应双脚来回移动（例如，如按下油门踏板上用双脚）。当屏幕是空白的，身体要完全放松。 </li><li>在运行过程中，每一个身体的一部分是重复20次。理想的情况下，应该有100个数据点，这意味着应该有一个5运行的总。多个会话，更少的运行是必要的，因为这个问题是能够更好地执行可想而知任务。 </li></ol></li><li>脑电图特征分析<ol><li>为了确定主体能够自愿调节，以控制的BCI脑电图特点，校准数据分析脱机状态下使用BCI2000离线分析工具，包括与BCI2000。此工具收集到的数据转换成频域FE结温，这表明在不同的动作改变，并最大限度地与任务相关的频率和位置。这些功能可以被用来在BCI实验。 </li><li>要确定应使用哪些功能，启动BCI2000离线分析工具。 BCI2000包括一个广泛使用的分析工具，应更多信息咨询教程。 </li><li>确定脑电图特点是强烈的每个动作相关，通过查找分析工具产生的田块大的R -平方值。最大的R -平方值（例如，大于0.2）的渠道和频率箱，然后可以选择作为一个特定的方向控制信号组件。例如，功能，右手条件的变化，应设立光标移动到屏幕右侧。 </li><li>同样重要的是，要记住，减少亩/β节律在与一个关联的运动幅度。因此，为了将光标移动到右边，正x方向，这个功能应该有一个与它相关的负重量。 </li><li>选择的渠道和频率应与已知属性的皮质感觉运动节律一致。也就是说，相应的重大变化与想象的右手运动应该看到较对侧（左），C3和CP3附近运动皮层和8-12赫兹和/或18-28赫兹附近为中心。同样，左手运动应在C4和CP4电极右运动皮质上的变化结果，和脚的动作应该会出现比CZ和CPZ。如果这些位置和值是不同的的，那么它可能是其他一些噪声或随机效应测量，不应该作为控制功能配置。 </li><li>对于每个条件，四个最大的R -平方值应选择的通道数量和BIN号。的频率2赫兹箱，所以被安排在高R平方10-12赫兹的功能将出现在6斌。有了这些值，系统可以配置为光标控制任务。 </li></ol></li><li>在线反馈会话配置<ol><li>配置BCI2000启动光标移动会议。 </li><li>在实验开始之前，有几个参数需要配置。首先，空间滤波器应配置一个共同的平均参考。要做到这一点，按Config中的BCI2000运营商带来了设置列表，并按下"筛选"选项卡。 </li><li> SpatialFiltering，和改变SpatialFilterType下拉框，以便它说，"共同平均参考（CAR）。" </li><li>根据SpatialFilter汽车产量，列表中的频道名称或所选号码在校准会议。例如，您可以键入"C3 CP3 C4的CP4锆CPZ"（不带引号），并BCI2000会知道使用哪条通道（通道标签，在"源"选项卡下的"通道名称"字段中列出的）。 </li><li>其次，分类矩阵必须配置使用选择功能。在"筛选"选项卡，分类参数，然后按编辑矩阵。 </li><li>应列数设置为4，行数，应等于所选功能的总数。每个矩阵的行对应到个别的功能。 </li><li>第一列应该包含所有正在使用的通道名称，如C3，C4等，第二列选择控制箱。很可能进入一个bin号码或特定的频率，进入"6"或"11Hz"，将选择同斌，BinWidth参数"下的"源"选项卡"2赫兹。第三列是输出通道;的值"1"对应的水平运动，"2"对应的垂直运动。应设置为1通道C3，CP3，C4，和CP4水平的光标控制任务; C3，CP3，C4，CP4，CZ和CPZ应设置一个垂直的光标控制任务2。最后，第四列拥有的功能，重量，并应符合既定的方向相反 ，例如，C3和CP3应加权-1向右移动光标，和C4和CP4应加权使其左右移动。向下移动光标，CZ和CPZ应加权1，向上移动光标，C3和C4应加权-1。 </li></ol></li><li>光标移动任务<ol><li>现在，该系统是用正确的设置配置，它是时间开​​始的实验任务。 </li><li>该系统配置，四个目标之一，这样就会出现在审判。该课题的目标是将光标移动到正确的目标，使用相应所需的运动方向（例如，右手向右移动，双脚向下移动等）想象运动。 </li><li>对于第一次试验中，将光标约束轴的目标。也就是说，如果目标是在顶部或底部，它是只能移动光标向上或向下，如果这是在左或右的画面，光标只能向左或向右移动。 </li><li>当开始运行时，字母"T"出现在2秒钟的画面。下一步的目标之一出现1秒。 </li><li>在此之后1秒的时间内，光标出现在屏幕中间。主体采用适当的想象运动直接目标的光标。如果这个问题击中目标，它会改变颜色。否则，受5秒前打次会议的目标和计小姐。 </li><li>庭审结束后​​，有2秒审间的间隔，在此期间，这个问题可以放松，眨眼，吞咽，或以其他方式重新调整职位。在试验中，运动应保持在绝对最低，以减少运动伪影或肌肉文物。这也有利于在一个昏暗的房间，舒适的椅子坐。 </li><li>经过20次试验，BCI2000进入暂停状态。在这段时间内，它可能需要重新调整一些设置，如果这个问题是无法将光标移动。 </li><li>如果4运行后的主体仍是无法volitionally移动光标，可能有必要重新分析BCI2000离线分析工具收集的数据。选择基础上的新功能地块的新途径和新频率。它可能需要多次运行或可能的几个会议前一个主题能够成为精通的任务。 </li></ol></li></ol>第6部分:代表性的成果: <ol><li>图1，图2显示了R -平方值的校准程序和头皮地形，应选择光标控制渠道和频率箱。 </li><li>一个训练有素的主体应该是1或2秒内能够快速移动光标移动到如图所示的目标。 </li></ol><img src="/files/ftp_upload/1319/1319fig1.jpg" alt="图1" /> 图1 A）和B）在10-12赫兹波段的光谱变化，在现实和想象的动作用右手中的地形。三）在休息C3上的功率谱（虚线）和运动（固体）。 D）R -平方的运动过程中的权力相比，休息。 <img src="/files/ftp_upload/1319/1319fig2.jpg" alt="图2" /> 图2中的R -平方跨越想象右手运动的所有通道。 X轴是在2赫兹箱从0到70赫兹的频率。 Y轴是通道号。最高的R -平方值发现渠道，9，10，17，18和19，其中包括对侧手运动皮层区。

<ol>
	<li>Fabiani, G. E., McFarland, D. J., Wolpaw, J. R., Pfurtscheller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+eeg+activity+into+cursor+movement+by+a+brain-computer+interface+(bci).">Conversion of eeg activity into cursor movement by a brain-computer interface (bci).</a> IEEE transactions on neural systems and rehabilitation engineering. 12 (3), 331-338 (2004).</li><li>Wolpaw, J. R., McFarland, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+two-dimensional+movement+signal+by+a+noninvasive+brain-computer+interface+in+humans.">Control of a two-dimensional movement signal by a noninvasive brain-computer interface in humans.</a> Proc Natl Acad Sci USA. 101 (51), 17849-17854 (2004).</li><li>Schalk, G., McFarland, D. J., Hinterberger, T., Birbaumer, N., Wolpaw, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bci2000:+a+general-purpose+brain-computer+interface+(BCI)+system.">Bci2000: a general-purpose brain-computer interface (BCI) system.</a> IEEE transactions on bio-medical engineering. 51 (6), 1034-1043 (2004).</li></ol>

<ol><li>至关重要的是，电极阻抗低，但没有太多的凝胶用来降低阻抗。一个单一坏通道，可以影响所有的人，通过共同的平均参考。如果阻抗可以不被多次尝试后减少，建议，一个快速的插入电极可用于，这可以简单地将进入的通过孔坏电极针放在通过注射凝胶，并在录音插入。 </li><li>在第一届会议，主题可能很难想象所需的动作。在这种情况下，可能会有所帮助他们做出真正的动作，第一，真正的运动数据进行?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>BCI2000- Compatible Amplifer System</td>
<td>&nbsp;</td>
<td>g.USBamp</td>
<td>&nbsp;</td>
<td><a href="http://www.gtect.at">http://www.gtect.at</a></td>
</tr>
<tr>
<td>BCI2000- Compatible Amplifer System</td>
<td>&nbsp;</td>
<td>Tucker-Davis Technology</td>
<td>Rx5 or Rx 7</td>
<td><a href="http://www.tdt.com">http://www.tdt.com</a></td>
</tr>
<tr>
<td>EEG cap</td>
<td>&nbsp;</td>
<td>Electro-cap International</td>
<td>&nbsp;</td>
<td><a href="http://www.electro-cap.com">http://www.electro-cap.com</a> At a minimum, the cap should have electrodes over hand and feet areas (C3, C4, and Cz). Additional channels can be used for control (CP3, CP4, CPz) and for spatial filtering as well, which will improve the signal quality.</td>
</tr>
<tr>
<td>Conductive gel</td>
<td>&nbsp;</td>
<td>Electro-cap International</td>
<td>&nbsp;</td>
<td><a href="http://www.electro-cap.com">http://www.electro-cap.com</a></td>
</tr>
<tr>
<td>PC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Running Windows XP or Vista (at least Pentium 4, 2 GHz, 1 GB RAM)</td>
</tr>
<tr>
<td>Two monitors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Each at least 19in (one for the subject and one for the researcher)</td>
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using an eeg-based brain-computer interface for virtual cursor movement with bci2000

一个脑 - 机接口（BCI）的功能，把一个神经信号的脑电图（EEG），成一个，可以用来控制计算机或其他设备的信号，如。在选定的频率垃圾箱的脑电信号的幅度进行测量，并翻译成一个设备命令在这种情况下，计算机光标的水平和垂直速度。一，脑电图电极应用到用户的头皮，使用记录大脑活动的第。接下来，校准程序是用来寻找脑电图电极和功能，用户将学习自愿调节使用的BCI。在人类中，在万亩功率（8-12赫兹）和β（18-28赫兹）频段下降幅度在一个真实或想象的的运动。这些变化可在脑电图实时检测，并用来控制BCI（[1] [2]）。因此，在筛选试验中，用户要求，使几种不同的想象与他们的手和脚的动作，以确定独特的脑电图改变与想象的运动功能。从这个校准结果将显示使用的最佳渠道，这是配置，以便在毛乌素和β频段的振幅变化垂直或水平移动光标。在这个实验中，通用的BCI系统BCI2000是用来控制信号采集，信号处理，并反馈给用户[3]。

Watch this Scientific Journal Video about Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000 at JoVE.com

Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000

在这个视频中，我们展示了一个脑 - 机接口实验，包括设立脑电图帽，校准系统，并培训用户，移动光标在两个尺寸，用想象运动一个需要运行的步骤。

基于EEG的脑机接口使用虚拟光标移动与BCI2000

A brain-computer interface (BCI) functions by translating a neural signal, such as the electroencephalogram (EEG), into a signal that can be used to ...

using-an-eeg-based-brain-computer-interface-for-virtual-cursor

Research

JoVE Journal

Biology

17.6K 次观看.  University of Wisconsin-Madison. 在这个视频中，我们展示了一个脑 - 机接口实验，包括设立脑电图帽，校准系统，并培训用户，移动光标在两个尺寸，用想象运动一个需要运行的步骤。

视频: Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

科研

生物学

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

在鼠年达到熟练的前肢结构:一个运动评定量表

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

在临床的虚拟现实使用生物反馈:勇敢的项目

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Nanopodia  - 薄，易碎膜预测与细胞运动和细胞间的相互作用角色

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

从人类肌肉提比利斯前部高级隔间收集骨骼肌肉活检，用于机械评估

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...