在这一研究的微反射镜装置是由惠特克基金会授予RG - 02 - 0682到N Boustany。正在进行的工作是由美国NSF - DBI - 0852857授予为N Boustany。马币帕斯特纳克是部分支持由罗格斯大学总统的研究生奖学金。我们还要感谢在我们的研究和博士的DN Metaxas用于有益的讨论有关光学过滤策略iBMK细胞大肠杆菌白色博士。

1。获得细胞的准备<ol><li>细胞接种前一天需要Mitotracker绿色标记的线粒体荧光成像。 </li><li>删除100μm的原液Mitotracker绿色在DMSO以前从4 ° C冰箱，和温暖的手到室温。还拿出牛主动脉内皮细胞（BAEC）细胞培养液中也事先准备好的暖到37℃，在台式水浴。 </li><li> Mitotracker和培养基一旦回暖，这些一定要消毒，戴手套的手和所有的外表面用70％乙醇溶液的容器放入引擎盖。不要打开引擎盖上的光，荧光标记，对光敏感，迅速将光漂白在室温光。 </li><li>线粒体标签正确的浓度是非常重要的。不会太少标签线粒体有效，而过多的Mitotracker毒性作用。一个孵育45分钟内与细胞浓度为100纳米的Mitotracker效果很好。准备10毫升培养液15毫升管加入100μLMitotracker股票浓度。这将使大量至少有一项实验。 </li><li>真空线连接到一个巴斯德吸管吸出旧媒介与标记介质更换现有的媒介。然后立即在6孔板，加入2毫升的标记介质​​以及每个占领文化。 </li><li>由于荧光标记是对光线敏感，在孵化器的替换细胞迅速直接室内光线不暴露。这个占地6孔板用手中的行之有效的。这些细胞会留在孵化时间为45分钟。 </li></ol> 2。获取的光学装置准备就绪<ol><li>虽然细胞在等待孵化器，我们已经打开的光学装置。在光学室，第一次打开汞弧灯，计算机，显微镜，摄像机，激光。然后在数字微镜器件（DMD）和纺纱扩散插件。 </li><li>检查，以确保光发射是通过显微镜目镜，以确保视野明亮的激光灯照亮对齐。 </li><li>清洁一件镜头纸折叠成一紧平方米的目标，并用止血钳紧紧把握。成氨的玻璃清洗液浸的镜头纸进纸吸收一个很小的数额。 RAP的止血钳在你放手几次删除任何多余的。用跨越目标，坚决申请一个干净，连续刷卡，从一端到其他，在镜头中间的目标。不要再刷卡或擦洗。丢弃使用过的纸张。 </li><li>要加载的样品，放置在63X油浸物镜下降的目标1-2小滴浸油，而目标是一路下滑的刻度。然后放置在舞台上的刻度。然后提高的目的，使油"争夺"的样本。重点在目镜的样本。 </li><li>要对齐冷凝器，调整冷凝器的高度，它是在中央科勒照明集中冷凝器领域停止的六角形边缘对齐。中心的冷凝器领域停止，如有必要的领域，通过转动聚光器旋钮。冷凝器光圈应该被关闭。 </li><li>开始IPlab方案和输入设置操作RoperScientific级联512相机。确认相机设置帧传输模式。启动运行"获得焦点"命令实时预览。设置索引前缀和文件的位置，该图像将被保存。 </li><li>开始RSImage方案和输入设置操作的CoolSnap程序。时钟模式应设置为正常。 </li><li>启动DMD软件和地方的脚本菜单上的暗场光圈，由"负载"和"复位"命令，并运行脚本。 </li><li>通过设置optovar和视显微镜LSM的DMD和Cascade 512相机的光发送。这将发送通过DMD和对齐光学图像投射到"防治荒漠化公约"的DF形象。暗场（DF）的图像应该出现在实时预览在IPlab已经展开。调整显微镜的微调对焦，如果有必要侧重于实时预览图像。 </li><li>采取的角度，用"收购单"命令领域的快照。设置足够高的曝光时间，以确保至少为10000计数的图像信号。收购完成后，使用"保存为索引"命令，将图像保存到磁盘。这种形象的刻度措施领域的视角（FOV）的大小。 </li><li>现在，移动的刻度样品的刻度，使超出FOV是如此，只有背景是可见的。在收购了该领域的背景图像足够长的曝光时间，以确保获得至少5000计数信号。此图片将有助于在未经过滤的图像的背景减法。 </li></ol> 3。载入滤波器和使用收购过滤背景图片设置<ol><li>现在，我们需要掌握的Gabor滤波的背景图像。 Gabor滤波器银行脚本加载到DMD控制软件。运行整个脚本缓冲过滤器的DMD的板载内存，这可能需要几分钟。 </li><li>一旦整个脚本缓冲，我们现在可以获取过滤图像的背景。使用的启动和停止内DMD软件的标记，以指示DMD的负荷只有一个对应时间过滤器的Gabor -的过滤器，并运行该脚本。该过滤器过滤后的图像，实时图像预览，应改变从暗场。 </li><li>从磁盘，在IPlab打开的收购脚本。调整曝光时间，以确保至少2000信号的罪名是被收购。由于DMD脚本运行，取消在IPlab实时预览，并运行收购脚本。这将自动获取，索引，过滤后的图像保存到磁盘。 </li><li>第一形象一旦被收购，停止在DMD软件上运行的脚本，并删除​​从脚本中使用的命令。替换下一个过滤器集的开头和结尾的启动和停止标记。重复IPlab收购。 </li><li>重复步骤3.4，直到整个滤波器已使用，并已经获得并保存所有过滤图像。 </li></ol> 4。电镀细胞<ol><li>截至目前，细胞很快就会准备好实验板。在实验室台式烙铁的插头。删除L15查看介质和热至37 ° C。请用纸巾和Kimwipe工作站。撕裂和捻转Kimwipes灯芯。灯芯将帮助转移流体和单元板。 </li><li>在此之后，我们需要板块的样本。我们使用的加工的金属样品架板我们的细胞，使"盖玻片三明治"与金属板之间。从上部外围的金属板孔每边的凹槽两端延伸大约一半左右的注射器应用的真空润滑脂薄珠。轻轻按下一个干净的没有。 1盖玻片上的油脂。翻转板和申请孔周围的油脂。关掉房间的灯。 </li><li>现在，我们得到的细胞，从孵化器处理用70％乙醇消毒手套丁腈考试的孵化器中的内容。从孵化器中取出电池板，屏住呼吸，而孵化器的大门是敞开的的。要小心，以尽量减少室内光线的曝光。 </li><li>取出盖玻片，将用于从6孔板实验指出，在面对良好的一方是与细胞贴面。仔细干燥的盖玻片上，双方直到它几乎是完全干燥，同时保持跟踪盖玻片一侧的细胞。然后按盖玻片，细胞的一面向下，到在观看孔润滑的金属板，使得确保没有空气间隙内的油脂层。油脂必须形成一个防水密封，让我们加载的L15培养基的细胞。一旦你一定是这个，翻转板背面超过。 </li><li>移液器的L15培养基上盖玻片和金属板之间的凹槽的液体，迫使通过镀细胞。一次移液200μL的效果很好。首卷吸，应填写与液体几乎延伸到另一边的凹槽的盖玻片之间的空间。 </li><li>另一个镀细胞液200μL移液器，但这个时候，灯芯举行反对格罗夫使介质流向从一个侧面。这洗细胞，并删除旧媒介的任何痕迹。要小心，以防止任何气泡在液体中形成，在这一步。重复这个过程2-3次，每冲洗一个新的灯芯。 </li><li>还记得我们烙铁插入吗？现在它被使用的时间。倒挂再次翻转板，支持从盘的边缘，使药液被困在细胞内水库，不能往下淌。蘸到valap烧杯烙铁。这很快就会融化一些valap然后将保鲜烙铁。仔细申请使用（这是现在朝上），烙铁头作为撒施底部的盖玻片边缘周围的熔融valap。继续浸渍和应用，直到你去周围的盖玻片周边所有的方式，盖玻片密封金属板。 </li><li>底部的盖玻片细胞增长，并可能有残留物从干涸的介质暴露侧。从盖玻片表面残留清洁球一个Kimwipe和清理机克盖玻片在一个单一的滑动很像清洁客观的议案。这确保了在该中心，将被视为最干净的盖玻片。 </li><li>拔下烙铁和观察相同的遏制和无菌程序返回6孔板的孵化器。以镀细胞光学实验室和装载步骤2.4和2.5所述的目标。 </li></ol> 5。进行实验<ol><li>找到一个漂亮健康的细胞领域。 </li><li>收购视野的暗场图像。在微分干涉对比（DIC）的对齐显微镜和收购DIC的形象。确保曝光时间足够长，以确保信号是足够的。 </li><li>现在我们已经获得的其他相机上的荧光图像。要获得对CoolSnap DIC的图像，我们使用的蓝色LED连接到冷凝器，更换，并在必要时将其取出。虽然仍然在DIC显微镜对齐，发送光CoolSnap 1.0X和视显微镜optovar设置到100％双目。转移的形象，从目镜相机。将LED在冷凝器照亮的领域和预览RSimage视野，如果有必要，调整微调对焦。收购DIC的形象，并保存到磁盘。请注意如何视野是从来自级联的相机获得一个不同。这些图像将被登记在实验后的分析阶段。 </li><li>获得的荧光图像，调整过滤器的多维数据集的荧光filtercube。获得一个简要转折点上使用的荧光显微镜的激发的形象，然后将其关闭，收购完成后尽快。由于我们集中在DIC样品，荧光图像，以及重点。这节省了荧光的暴露时间，从而减慢漂白。荧光图像保存到磁盘。 </li><li>现在我们已经获得过滤的图像。复位暗场显微镜，并通过LSM端口重新发送2.9光。 </li><li>运行在3.3-3.5的整个的Gabor滤波器脚本。现在我们已经完成了一个时间点的数据采集。 </li></ol> 6。切换媒介揭露细胞星形孢菌素（STS）的，和维护整个实验中<ol><li>虽然细胞仍然在舞台上，而不会干扰视野，转出常规的L - 15中包含一个解决方案，从4毫米的STS的股票在DMSO溶液中作出的STS 1微米。使用排汗的方法步骤4.6中所述的开关媒体。 </li><li>现在，我们重复步骤5.2-5.6随后的时间点。我们重复这个过程直到完成实验。 </li><li>在实验过程中，必须加入更多的媒体将这样的样本不变干。这是通过吹打成细胞板的小树林，没有从舞台中取出，而不会干扰视野的媒介。 </li></ol> 7。代表性的成果在实验结束时，收集到的数据将包括大量的过滤提取的亚细胞结构的数据，需要处理的图像。两个例子表明光学过滤器9的Gabor过滤器，过滤期间，S =0.95μm，高斯信封标准差s = S / 2 =0.45μm的，和方向Φ= 0 °〜Φ= 160 ° 20组成银行度增量。 （更多细节见[1]）。 例1:海洋硅藻我们首先应用于我们的方向敏感的过滤银行，海洋硅藻样品（北卡罗来纳州生物供应公司）与导向功能，在暗视野（DF）的成像（图1）清晰可见。光学过滤的图像显示旁边的样品未经过滤的图像进行比较。 <img src="/files/ftp_upload/1915/1915fig1.jpg" alt="图1" /> 图1:暗视野（DF）和光学过滤海洋硅藻的形象 。 在右下方的图像（在最左边的面板上的白色箭头），我们将分析的硅藻。 九个图像的Gabor滤波的硅藻处理为对象的方向和圆度的像素像素。加工包括（1）在每个像素中总结的九个所有的Gabor滤波的图像测量的响应，从而编码响应意义的信号响应，以确定总体规模，和（2）寻找Gabor滤波器的定位，Φ，在响应最大化，这个最大反应的比例，从而编码在何种程度上在每个像素中的对象有择优取向的角度平均响应。取向的程度是密切相关的粒子的几何宽高比。在图。 2B，THE的像素的整体响应滤波器组（参数1）的方向或长宽比（参数2）程度进行编码，分别在色彩饱和度和色相。近1长宽比（蓝色）是目前在这方面有没有首选的响应角度，而更大的价值（红色），表明在哪些领域存在较高的首选的角度响应。下部结构粒子的方向是在颤动情节（图2C），其中每行紧密合作，同意与当地的基本面向对象的可见未经过滤的暗场（图2A）编码。 <img src="/files/ftp_upload/1915/1915fig2.jpg" alt="图2" /> 图2:答:硅藻的暗场图像 。 乙:面向对象的图像。色阶表示取向度（长宽比），而亮度编码的总的Gabor滤波器响应意义。 C:反应强度≥10％的最大的对象定位 。 线段表示相应的结构的长轴。 例2:细胞凋亡在这里，我们展示硅藻同样的方式处理的星形孢菌素（STS）处理的牛内皮细胞过滤的图像。图3显示了一个未经过滤的暗场（DF）的细胞随着九个过滤图像在时间T = 180分钟的图像。前STS处理。 <img src="/files/ftp_upload/1915/1915fig3.jpg" alt="图3" /> 图3:暗视野（DF）和光学过滤的一个领域包含几个生活内皮细胞的图像。 过滤后的图像随后被收购了STS处理后三小时内每20分钟一班。图4a显示宽高比地图作为时间函数的细胞。在这种情况下，颜色的色调代表的取向度（标记orientedness）为图的颜色的色调。 2B以上。然而，长宽比的亮度是不加权平均滤波器响应。通过注册与我们在这些细胞中的线粒体标记（图4b）的荧光图像的宽高比地图，我们决定，测量宽高比下降局限于细胞含有线粒体的地区，随之而来的是与线粒体碎片可以直接观察到在同一细胞的荧光图像。图5显示时间图，描绘了在长宽比的时间在发生凋亡的细胞功能变化。在每个单元中，有一个纵横比下降在T = 60-100分钟注册用荧光线粒体的地区，但在昏暗的背景荧光地区注册的地区。 <img src="/files/ftp_upload/1915/1915fig4.jpg" alt="图4" /> 图4:高宽比（a）和荧光（B）与凋亡诱导剂，星形孢菌素处理的内皮细胞的图像。 <img src="/files/ftp_upload/1915/1915fig5.jpg" alt="图5" /> 图5:时间图比较粒子长宽比（orientedness）在血管内皮细胞与星形孢菌素治疗减少。单独的轨迹代表单个细胞内的时间图 。 在orientedness下降局限于区域的细胞，注册用荧光线粒体（左图）和缺席余下的背 ​​景荧光地区（右图） 。 现在，我们已经确定的长宽比下降对应的线粒体的分裂，我们可以在这些细胞中诱导凋亡，用我们的光散射法测量，而无需标签的细胞碎片，并研究不同的遗传和实验条件，在此影响动态。

<ol>
	<li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., White, E., Boustany, N. N. <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+Subcellular+Texture+by+Optical+Gabor-Like+Filtering+with+a+Digitial+Micromirror+Device.">Measurement of Subcellular Texture by Optical Gabor-Like Filtering with a Digitial Micromirror Device.</a> Optics Letters. 33 (19), 2209-2211 (2008).</li><li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+size+discrimination+of+submicron+objects+using+optical+Fourier+filtering+based+on+two-dimensional+Gabor+filters.">Highly sensitive size discrimination of submicron objects using optical Fourier filtering based on two-dimensional Gabor filters.</a> Optics Express. 17 (14), 12001-12012 (2009).</li><li>Zheng, J. -. Y., Pasternack, R. M., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+scatter+imaging+with+a+digital+micromirror+device.">Optical scatter imaging with a digital micromirror device.</a> Optics Express. 17 (22), 20401-20414 (2009).</li></ol>

的方法在某些方面已包括在发明披露，美国罗格斯大学文案＃09-049。

上面介绍的方法对象的编码粒子的大小或方向，例如产量形态地图。这种结构上的信息可用于在几个方面: <ol><li>它可以用来作为一个初始画面，以确定改变在一个特定的治疗，然后再进一步分析这些地区与特定的分子和生化分析的组织或细胞的地区。 </li><li>它可用于荧光的结合，产生的细胞，它结合了定量表征的亚细胞结构（用这种方法）的分子活动的同时理解（荧光）多式联运分析。 </li><...

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<td>DMEM</td>
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<td>Invitrogen</td>
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<td>Low glucose DMEM</td>
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<td>Liebowitz L15 medium</td>
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<td>Invitrogen</td>
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<td>Without phenol red</td>
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<td>L-glutamine</td>
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<td>Invitrogen</td>
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<td>Mitotracker Green</td>
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<td>Invitrogen</td>
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<td>Bovine Brain Extract</td>
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<td>Clonetics</td>
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<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gemini Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Staurosporine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dymethylsulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>Axiovert 200M</td>
<td>&nbsp;</td>
<tr>
<td>DMD</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>TI 0.7 XGA DMD 1100</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roper Scientific</td>
<td>Cascase 512B</td>
<td>High (16 bit) dynamic range CCD</td>
</tr>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roped Scientific </td>
<td>Coolsnap cf</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

optical scatter microscopy based on two-dimensional gabor filters

我们展示一个微小的仪器，可以测量未染色的活细胞内细胞器的形态和组织所产生的亚细胞质地。拟议文书的扩展的无标记的光学显微镜的细胞器的大小和形状的纳米级变化的敏感性，可用于加速程序性细胞死亡或细胞的细胞器官动力，底层的基本生物过程有关的结构与功能关系的研究，如，分化。显微镜可以很容易地在现有的显微镜平台上实施，因此，可以传播到各个实验室，科学家可以实现和使用不受限制地访问所提出的方法。 提出的技术能够通过二维光学Gabor滤波器，通过观察细胞的亚细胞结构的特点。这些过滤器可以进行调节，以纳米（10纳米的）的灵敏度，非球面细胞内细胞器的大小和方向有关的具体形态属性感。虽然弹性散射产生对比的基础上，不依靠技术进行了详细的逆散射米氏理论模型或提取形态测量。因此，这种技术适用于非球面的一个精确的理论散射描述是不容易的细胞器，并提供了独特的形态参数，可以在未染色的活细胞内获得评估其功能。与数字图像处理，因为它直接工作对象的字段比离散对象的强度，而改造相比，该技术是有利的。它不依赖于高图像的采样率，因此，可以用来迅速屏幕在一段时间内数以百计的细胞形态的活动，从而大大方便了超越个人的细胞器荧光共聚焦显微镜高倍放大的数字图像的分割和重建的细胞器结构的研究有限领域。 在这个演示中，我们从海洋硅藻显示的数据来说明的方法。我们初步数据还显示，从收集到的活细胞如何使用该方法可应用在相关的生物背景下的思想。

Watch this Scientific Journal Video about Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters at JoVE.com

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

我们表现​​出一个暗场显微镜基于Gabor般的过滤来测量单个活细胞内的亚细胞动力学的方法。该技术是敏感的细胞器结构的改变，如线粒体碎片，。

基于二维Gabor滤波器的光散射显微镜

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

optical-scatter-microscopy-based-on-two-dimensional-gabor-filters

Research

JoVE Journal

Biology

9.5K 次观看.  Rutgers University. 我们表现​​出一个暗场显微镜基于Gabor般的过滤来测量单个活细胞内的亚细胞动力学的方法。该技术是敏感的细胞器结构的改变，如线粒体碎片，。

视频: Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...