作者衷心感谢布兰登J. Zipp和肯B.卡普兰在这项研究中所使用的酵母菌株提供访问。作者还感谢胡里奥列宁多明格斯，拉米雷斯与摄录援助。高考承认授予数量5RC1GM91755从美国国立卫生研究院的资金支持。

1。制造和装配<ol><li>广场内的22.96厘米（9"）馅饼泛泛一边约2厘米，如图1c所示，使用一个黑色的记号笔，铝散热器的标志孔注铝散热器。铝块（在本地废旧金属店或在网上购买<a href="http://www.onlinemetals.com">http://www.onlinemetals.com</a> ）不与预钻孔，您将需要安排有洞钻，埋头前第1步，以确保铝块馅饼泛，我们采​​用5孔，以确保块平台和4个额外的孔，让氮气泡沫从铝下逃生。 </li><li>广场的铝质散热片的位置附近的冷冻格框和马克的缺口，每个侧面。 </li><li>每个标记的位置，使用＃17钻铝散热器安装孔位＃35钻头和冷冻格箱孔钻导向孔。 </li><li>摩的铝质散热片，用5 - ＃10，1.9厘米（3 / 4"）的平头开槽螺栓和相应的螺母，以及15 - 10号垫圈放置两个垫圈下方的铝块和第三的背面。 。馅饼泛每孔注:观赏区附近的铝块的无抵押出挑可以导致振动限制成像能力的阶段检查，以确保所有的安装螺栓拧紧。 </li><li>从锅饼作为冷冻格箱装载的背面，插入3 - ＃4，0.95厘米（3 / 8"）轮开槽螺栓和相应的螺母。 </li><li>磁带4筷子之上另一个用筷子对。磁带每对相对边缘的蛋糕盘作为垫片。 </li><li>湿的蛋糕和馅饼的顶部和底部表面盘分别与DDH 2 O 。 </li><li>两层绝缘喷涂泡沫填充蛋糕盘。润湿前准备进行下一个步骤，每一层。 </li><li>按泡沫的馅饼泛。 </li><li>倒挂的平底锅翻转，蛋糕上的泛按，直到垫片接触的馅饼盘。放置任何上盖蛋糕盘的加权对象，让坐在一夜之间治愈。 </li><li>锯齿刀，切掉多余的干喷涂泡沫保温锅的边缘，取出筷子。 </li><li>删除从馅饼盘蛋糕盘。铝散热器的馅饼泛现在是绝缘的，将被称为"低温阶段"（图1A）。 </li><li>剪贴板在低温阶段顶部放置一个透明的塑料（任何颜色和大于3毫米厚）。马克用黑色标记的冷冻格箱安装位置画一个圆圈，直径约1.5倍的单轮冷冻格框。剪出一个1.9厘米（3 / 4"）的孔，孔锯剪贴板将称为"载入画面"（图1b）。 </li><li>之上直属物镜与散热器的低温阶段，并在显微镜放在一个新的透明剪贴板。一个黑色的标记，标记的目标的路径，因为它们旋转。 </li><li>剪切清除剪贴板的边缘半圈沿标记线。这可以完成拼图或使用一个7.62厘米（3"）孔锯多次在视频所示。 </li><li>最后远离了半圈，但仍然菜面积内切一个1.9厘米的洞（3 / 4"），这将是该端口加气站液氮（LN 2）如果水平下降，而成像，这将被称为剪贴板"查看屏幕"（图1C）。 </li></ol> 2。样品制备<ol><li>淡化对数期酵母细胞合成完整的（SC）的媒体发展到一个适当的浓度为1 × 10 6细胞/毫升在水中。 </li><li> 2毫升稀释后的酵母吸管到R2 / 1 400目多孔碳包覆铜冷冻电镜网格（SPI用品，西切斯特，PA），并允许15秒吸附。 </li><li>通过触摸发车2秒（Kimwipe）的软纤维组织撕裂一块远离电网的表面吸干多余的液体。 </li><li>如果有必要，电网的可洗3μL的水珠（1-3倍），而恶人在第3步，从后面的印迹。 </li><li>之前的最后一次洗涤，样品装入气动暴跌冷冻和挂网冲在步骤2.4。印迹用纸巾撕裂（Kimwipe），以去除表面的水大部分电网后，电网陷入液氮冷却的液态乙烷和转移到冷冻格框为储存 1注 :重要的是要表面上留下一层液体，保持样品的水分尽可能薄。不适当的印迹，将干出来的样品，或离开冰是不透明的电子束。 </li></ol> 3。低温光显微镜<ol><li>填写的低温阶段，LN 2，并迅速覆盖载入画面（单1.9厘米（3 / 4"）孔的塑料剪贴板） 。 </li><li>一旦金属达到液氮温度，通过1.9厘米（3 / 4"）加载屏幕孔，并安装到转移部分A5所描述的3个螺丝创建传输的冷冻格框。拧下的冷冻格框其持有人持有，以提高访问和删除，保持在3.10步之前单独杜瓦液氮下的冷冻格框持有。冷冻格框也应根据液氮保存，以防止样品电网变暖。 </li><li> 。笔芯在低温级的铝质散热片的顶部略低于第 2注 :LN 2的水平，必须定期检查，并在成像过程中进行重新充装，以确保LN 2与散热片接触 ​​不断。在一般情况下，加气发生在观看屏幕每隔10分钟通过填写端口。 </li><li>预冷的镊子尖2号法律公告，然后转移到平 ​​坦的观赏面的铝质散热片用镊子您的网格（S），使用1.9厘米（3 / 4"）加载屏幕孔。 </li><li>小心移动的低温阶段下的反射光显微镜的客观光圈。 </li><li>将透明上盖的载入画面观看屏幕。观看屏幕注意下方滑动，然后慢慢松开载入画面 :在这一点上，样品是最脆弱的。显微镜周围所有的空气电流应尽量减少，包括:呼吸，附近一开门，人走过去，等我们建议戴口罩，或挂一个透明的面罩，作为一项预防措施，在显微镜目镜以下。 </li><li>进入寒冷的气体环境中氮的低温阶段和扫描样品在平面上的散热器可视面积旋转的物镜。使用低倍率的客观标准的明场光学显微镜技术，重点对电网的中心，并采取概览图像。本实验所用的光学显微镜镜头的规格就列出了部分实验材料注 :LN 2不接触物镜和冷却的影响，我们还没有看到的图像退化或损坏的镜片。 </li><li>感兴趣的领域和重点在高放大倍率，明场，暗场，偏振光或荧光成像获取数据。可以肯定的感兴趣的区域的位置上记录的低倍率为未来相关的明场像。如果所有上面列出加气站LN 2的注意事项，然后从蒸发氮的正压力足以维持一个无污染的环境（独立室内湿度）的成像时间。 </li><li>完成后，取代加载屏幕，在观看屏幕的顶部滑动。删除放缓载入画面下方滑动，观看屏幕。 </li><li>从显微镜中删除的低温阶段，预冷的镊子，传输电网的冷冻格框。到冷冻格箱的冷冻格框支架螺丝密封对环境和存储和未来成像的持有人转移到液氮杜瓦。 </li></ol> 4。低温电子显微镜<ol><li>标准和技术成熟之后，装入一个冷冻电镜转让持有的样品电网，同时保持在任何时候都液氮温度下的标本。 </li><li>透射电子显微镜样品电网插入冷冻人。 </li><li>在一个合适的低倍率查看（50 - 500X标称的放大倍率），发现样本电网的中心。 </li><li>使用以前收集的低倍率从步骤3.7冷冻LM数据，以确定电网的中央铜标签的相对取向（如在图2中所述），并确定相关利益的具体领域。 </li><li>继续显微镜对齐，低剂量的冷冻电镜成像和数据收集。 </li></ol> 5。代表性的成果低温阶段（图1A）是一种有效的方式来收集冷冻LM低温荧光显微镜及相关cryo-LM/cryo-EM分析数据。图2显示了如何在低和高放大倍率的冷冻LM图像的组合，使您可以构建参考地图，你直接在冷冻电镜的具体领域。由此产生的冷冻LM参考图（图2b）是利用冷冻电镜数据采集期间找到在图3所示的确切地区。 <img src="/files/ftp_upload/2909/2909fig1.jpg" alt="图1" /> 图1 低温级 ）的低温阶段的布局由一个挂载表示瓦特的冷冻格箱转移第i个白色箭头和铝散热器。网格转2号法律公告，从冷冻格框，直接放置在散热器的可视面积的黑色箭头表示 B）图片描绘了载入画面的低温阶段。载入画面中的孔是直接放置在冷冻格箱安装和行为，作为一个港口转移样品和样品散热器用镊子从冷冻格中移动样品可视面积 。 C）低温在观看屏幕的物镜下阶段的位置。观看屏幕上的特殊切口，使物镜容易进入的位置不移动的低温阶段摆动。在观看屏幕远离样本区的孔行为填写端口，以补充LN 2的水平，如果有必要 ，作为LN 2。 <img src="/files/ftp_upload/2909/2909fig2.jpg" alt="图2" /> 图2。 冷冻- LM导航相关研究。A）低倍率酵母细胞冷冻LM坚持一个样品电网。B）相同的图像（一）在高放大倍率的荧光图像感兴趣的领域覆盖标志着一个充满橙色多边形样本电网的中心。该中心由一组4个广场，有一个额外的金属片，并提出开放三。对于一个额外的金属片的三个广场，两对共享的长期和短期形成一个非对称中心轴的标签。左上角可以看出，在这种不对称的中心，是用来表示旋转的角度和电网之间的冷冻- LM和冷冻电镜的霸道。从一个低倍率的形象感兴趣的许多领域可以被标记，并用来作为参考图冷冻电镜定位在相同的地区。C）放大的荧光冷冻LM感兴趣的区域的图像（b）中。 HTA1 - CFP，C -末端CFP组蛋白标记，可以看到一个绿色的点状结构标记细胞核的位置。d）对相应区域的冷冻电镜图像（C）。标尺代表 50微米。 <img src="/files/ftp_upload/2909/2909fig3.jpg" alt="图3" /> 图3。 冷冻- LM和冷冻电镜的相关性。领域的观点，描绘了相同的酵母细胞相关冷冻明场（A，D），低温荧光（B，E），和冷冻电镜（C，F ）。 比例尺代表5微米。

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作者声明没有竞争的金融利益。

<p class="jove_content"&gt;使用我们的低温阶段和样品制备技术，低温- LM和冷冻电镜之间的相关性研究，展览，比传统的室温方法，包括但不限于以下几个好处:快速固定<sup&gt; 1,2</sup&gt;，减少漂白<sup&gt; 3</sup&gt;和前冷冻电镜扫描样品的完整性<sup&gt; 4,5</sup&gt;或化学固定/嵌入<sup&gt; 6</sup&gt;。冷冻电镜数据采集的瓶颈之一是为转移到样品的TEM和扫描网格，找到合适的地区形象所需的时间。随着冷冻LM快速的数据采集<sup&gt; 5</sup&gt;，寻找良好的细胞所用的时间可以大大减少。因此，我们可以节省时间和金钱，通过预扫描和映射冷冻LM的兴趣领域和缓解这个瓶颈。</p&gt;<p class="jove_content"&gt;更昂贵或复杂的低温阶段，可能会允许更高分辨率的低温LM成像。然而，这里编造的阶段是比许多应用绰绰有余。如果在文字和视频的谨慎进行冷冻LM成像和样品电网传输，完全无污染的成像可以完成。这种方法允许有直接的关系相同的细胞，细胞器或大分子复杂的荧光灯和电子显微镜数据分散在碳支持或薄组织切片内中<sup&gt; 7</sup&gt;。应该指出，这种低温阶段的价值延伸到超越cryo-LM/cryo-EM的相关研究作为一个整体的光学显微镜领域。这种低温阶段是非常适合既微妙的样品和荧光团染料/污渍<sup&gt; 8</sup&gt;，量子点，或荧光标记的蛋白质<sup&gt; 9</sup可在室温化学与戊二醛和多聚甲醛固定和嵌入不稳定。因此，我们希望，这种低温阶段将提供更大的访问那些有兴趣在低温LM，先前已经由成本和没有足够的访问却步。</p&gt;<p class="jove_content"<em&gt;疑难解答低温光学显微镜</em</p&gt;<p class="jove_content"<em&gt; 1。样品吸附:</em&gt;不同的样品，可以表现出可变的吸附特性的碳支持。您可能会发现有必要使用其他类型的支持，如碳，多孔碳，或样本支持formvar涂电网电网。此外，如果样品是有约束力的电网，而不是碳表面的铜条的，它可能是必要的调整孵化电网的润湿剂或辉光放电前样品除碳的表面化学。</p&gt;<p class="jove_content"<em&gt; 2。舞台上的稳定性:</em&gt;如果您发现振动，它可能是由于超载重量，而不是LN2冒泡的样品阶段。的低温阶段增加的重量，有时会造成低频率振动来传输标准的3轴行程显微镜阶段，产生不利的影响通过模糊成像。这可以很容易地从下方支持在显微镜的旅游阶段，可调节的小型升降机或双螺杆武器可调适配器，如视频所示纠正。这些可调支持机制互动与非可移动部分的旅游阶段，因此仍然允许X和Y轴平移成像的要求。</p

特定的试剂和仪器表: <table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录＃ </th><th>评论</th><th>成本</th></tr><tr><td> 1 - 22.86厘米（9"）与倾斜边缘的馅饼泛</td><td>好厨师</td><td></td><td>当地的杂货店</td><td> $ 4.39 </td></tr><tr><td> 1 - 22.86厘米（9"）蛋糕盘</td><td>好厨师</td><td></td><td>当地的杂货店</td><td> 4.93美元</td></tr><tr><td> 4双筷子</td><td></td><td></td><td>当地的杂货店</td><td> $ .50 </td></tr><tr><td> 1 - 伟大的东西喷涂泡沫</td><td>伟大的东西</td><td></td><td>当地的五金商店</td><td> $ 5.98 </td></tr><tr><td> 1 - 4CM x8cm x1cm铝块</td><td></td><td></td><td>当地的五金商店或金属废料场</td><td> $ 10.00 </td></tr><tr><td> 5 - ＃10 1.91厘米（3 / 4"）的平头开槽螺栓/螺母</td><td></td><td></td><td>当地的五金商店</td><td> 0.98美元</td></tr><tr><td> 3 - ＃4 0.95厘米（3 / 8"）轮开槽螺栓/螺母</td><td></td><td></td><td>当地的五金商店</td><td> 0.98美元</td></tr><tr><td> 15 - 10号垫圈</td><td></td><td></td><td>当地的五金商店</td><td> 0.98美元</td></tr><tr><td> 2 - 透明塑料剪贴板</td><td></td><td></td><td>当地办事处商店</td><td> 6.84美元</td></tr><tr><td> 1 - 冷冻电镜格箱持有人</td><td>特德佩拉</td><td> 160-41 </td><td></td><td> N / A </td></tr><tr><td> 1 - 冷冻电镜网格处理杆</td><td>特德佩拉</td><td> 160-46 </td><td></td><td> N / A </td></tr><tr><td> 1 - R2 / 1多孔碳薄膜，400目铜冷冻电镜支持电网</td><td> SPI用品</td><td> 4340C - XA </td><td></td><td> N / A </td></tr></tbody></table>实验材料<ol><li> 酵母菌株:Hta1 CFP:根（KSC - 3382:从卡普兰实验室KanMX6:MATA，URA3 - 52，lys2 - 801 - 101 ADE2，TRP1 -Δ63，HIS3Δ200，LEU2Δ1，Hta1 - CFP:加州大学戴维斯分校）。 </li><li> 低温光学显微镜 ，低温光镜图像与徕卡DFC310 FX CCD相机徕卡DM4000M反映显微镜。明场，暗场和荧光图像被收购HCX PL FLUOTAR 5倍0.15 NA空气的目的，一个HCX PL FLUOTAR 10X 0.30 NA空气的目的，一个PL FLUOTAR 50X 0.55 NA空中目标或NPLAN 100X 0.75 NA空气的目的。明场和暗场图像，用一个12V 100W钨灯作为光源。 EL6000紫外线灯源的荧光，使用徕卡JC1（Ex.F.:BP三十〇分之四百八十〇，德国马克:唱片505，SF BP四十○分之五百三十五及唱片580）一起过滤立方。 </li><li> 低温透射电子显微镜:冷冻电镜图像被收购利用一个加坦626冷冻转让持有人（加坦，美国）在200千电子伏在JEM - 2100 - FEG的透射电子显微镜（JEOL，日本）操作系统。显微照片被记录在一个4096 x 4096像素的Tietz CCD相机（TVIPS，Gauting，德国）50，200，1200和4000 X标称的放大倍数。 </li></ol>

low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy

耦合低温光镜（冷冻LM）和低温电子显微镜（冷冻电镜）对细胞动力学的理解和超微结构的许多优点。首先，细胞可成像两种技术在不久的原生环境。其次，由于玻璃化过程中，样品保存快速物理固定，而不是缓慢的化学固定。第三，成像同一样品冷冻LM和冷冻电镜提供相关的数据从一个单细胞，而不是一个"有代表性的样品"的比较。虽然这些好处是从以前的研究，相关冷冻- LM和冷冻电镜仍然广泛使用有限的，由于购买或建立一个合适的低温光镜阶段的费用和复杂性。在这里，我们展示了大会，并用不到40美元，在当地的硬件和杂货店发现部分可以在任何实验室制造的一种廉价的低温阶段。这冷冻LM阶段的目的是反映装有长工作距离的空中目标的光学显微镜的使用。对于相关的冷冻LM和冷冻电镜研究，我们适应标本支持使用碳涂层标准的3毫米的冷冻电镜电网。网格吸附样品后，vitrifying样品和转让/处理电网先前建立的协议之后，允许多技术成像。因此，此设置允许任何一个反射光显微镜实验室有直接相关的冷冻水合样品成像。

Watch this Scientific Journal Video about Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy at JoVE.com

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

我们展示了一种低成本的低温阶段，设计，以适应最反射光显微镜的制造。这个实验室建成的低温阶段，实现高效，可靠的冷冻光线和低温电子显微镜之间的相关性成像。

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

low-cost-cryo-light-microscopy-stage-fabrication-for-correlated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

15.4K 次观看.  University of California Davis. 我们展示了一种低成本的低温阶段，设计，以适应最反射光显微镜的制造。这个实验室建成的低温阶段，实现高效，可靠的冷冻光线和低温电子显微镜之间的相关性成像。

视频: Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...