我们想感谢劳拉写的第一个版本的细胞装入协议和马库斯Goffau的和吉尔Zampar的为得分王线粒体形态Schippers。

1。硅片模具的生产和制备微流控芯片的创建软光刻模具生产的硅片。这些晶片可多次重复使用，以生产微芯片。可取的做法是执行各自的晶片的生产由一组专门在微流控6。 的晶片是由在两个步骤的光刻工艺，使用两种不同的负光致抗蚀剂层，SU-8 7。底部层是用来产生细胞俘获区（SU-8，2002;高度3-4微米），而频道与顶层（SU-8 2010，高度为10μm）。 Huang 等人 8可以发现在晶片生产过程中的印象。微流控芯片的图纸可以从作者的意见如何获得相应的晶片。 <ol><li>一旦获得晶片，切一块艰难的标签诠释ö薄带约0.5毫米，3毫米，用解剖刀，并仔细地将它们放置在晶片上稍微的信道结构的顶部之间的入口通道和micropad的阵列（ 图2A）。这将导致在一个大的附加信道，这将不仅可以用于在细胞装载过程中在每个芯片的形成，但同样重要的是微流体装置的稳定运行。 </li><li>用双层铝箔覆盖玻璃培养皿中，并把晶片的陪替氏培养皿中。铝箔将防止PDMS之间的晶片和陪替氏培养皿中运行。如果PDMS蠕变之间在晶片和玻璃培养皿中，晶片将成为永久地固定在陪替氏培养皿中。虽然晶片仍然是可用的，它不会是可能的，用以代替玻璃培养皿中，如果它打破，将需要被切出之后的PDMS。 </li></ol> 2。微流控芯片的生产<ol><li>将一个空塑料杯放在天平上，天平去皮。倒入40 PDMS克基地到塑料杯（12厘米直径的玻璃培养皿）。 PDMS固化剂添加用一次性吸管AW / W比为1:10， 即约4毫升。 </li><li>彻底的PDMS与固化剂混合几分钟。用一次性塑料移液管，用移液管的固化剂，这是可以做到。重要的是，该混合物充分混合，倒入晶片上之前。可怜的混合会导致不聚合的PDMS的一部分。 </li><li>倒入的PDMS在玻璃培养皿中的晶片的顶部。 </li><li> PDMS与固化剂的混合会导致气泡的形成中的PDMS混合物。这些气泡的PDMS聚合之前，需要被删除。的PDMS可浇注后晶片上的陪替氏培养皿放置在干燥器中连接到真空泵脱气。有了这个设置，它大约需要30分钟，从混合物中除去所有的气泡。 </li><li>聚合个ë键PDMS的陪替氏培养皿放置于120℃的热板的顶部与晶片1小时，然后在65℃下，一个小时。 </li><li>一起在晶片和聚合PDMS从玻璃培养皿中取出铝箔。剥下的铝箔和从晶片背面的薄的残余层的聚合PDMS。的PDMS层的晶片上，然后，可以从晶片上分离，小心地向上提起。 </li><li>将PDMS层倒挂（渠道朝上）在板凳上切出的单芯片设计印PDMS上用手术刀小心。尝试保留约3毫米的硅橡胶的边缘周围的通道，以改善芯片附着到玻璃盖稍后。 </li><li>打孔的PDMS中在Luer式短截线20线规推的PDMS的所有的方式通过在一条直线的方式（ 图2A）的入口，出口和侧流路的端部。 </li></ol>确保PDMS芯片仍然躺在倒在板凳上，而打孔的孔。这可以防止硅橡胶粘的内部的通道，这可能会导致堵塞的通道。 <ol start="9"><li>每孔冲孔后，删除该列的PDMS在鲁尔存根镊子之前再次拉动存根。 PDMS列否则可能会留下，并阻止刚打孔。 </li><li>清洁表面的灰尘颗粒和剩余的PDMS的芯片，通过将透明胶带就可以了，并立即再次取出磁带。同样，该芯片将被清洗的玻璃盖。 </li><li>将下面的UV灯，台式UV-臭氧清洗机的清洗芯片和玻璃盖。需要被粘合在一起的两侧必须面对灯泡。 </li><li>暴露的PDMS和盖玻片6-8分钟的UV光，然后直接把芯片放置在玻璃盖的暴露表面，在彼此的顶部。 </li><li>轻轻敲击左右两侧的芯片到促进附着的PDMS的玻璃盖，以消除任何气泡。不要拍打渠道结构的顶部，因为这可能会导致micropads得到重视，以及玻璃盖。 </li><li>将新制成的微流体设置在100℃的热板上60分钟。测试的PDMS芯片的边缘稍微抬起，玻璃盖​​和PDMS芯片之间的粘接强度。当无法解除从玻璃表面的PDMS嵌段，接合是成功的，该芯片已准备好使用。 </li></ol> 3。准备细胞装载芯片微流体装置的故障有可能导致介质泄漏到显微镜。为了避免这种情况，它是咨询使用的金属支架和/或密封的芯片的侧面的PDMS符合玻璃用指甲油或环氧胶。这可以防止介质泄漏的PDMS芯片的情况下，未结合不够好，玻璃盖。此外，管件连接，可以确保周围的插入点以类似的方式，以避免介质的泄漏。自制的水传感器，关闭注射泵在泄漏的情况下，可以作为一个额外的安全预防措施。附图的金属保持器，特别是设计用于在微流体芯片上，可以得到从作者。 <ol><li>放置在金属保持器的微流体芯片，硅凝胶的芯片和玻璃之间的保持器来创建一个防水密封的金属部分。轻轻拧紧螺母，没有打破玻璃。 </li><li>连接细管（ 约 5-15厘米长）侧通道和出口通道的芯片。镊子的使用，可以更容易地插入管的冲孔。 </li><li>填写一个50毫升的注射器鲁尔乐培养基，去除空气中的注射器，并把它连接顺序注射器过滤器，20号鲁尔存根，短而粗的管（ 约 2厘米长）和细管。确保薄浴缸e是足够长，以跨越注射泵和显微镜载物台之间的距离。 </li><li>将注射器在注射器泵，快进的细管的泵，直到完全充满介质。 </li><li>推到该芯片的入口通道连接到注射器的细管，让介质的流量为10微升/分钟（ 图2B）通过芯片。收集留下的芯片介质（ 例如，在培养皿中）。介质运行因为强韧标签大侧通道和出口通道的阻力之间的差异，通过侧通道。 </li><li>芯片放置在显微镜载物台和设置在焊盘上的焦点显微镜。 </li></ol>使用油浸目标是首选，因为油比水浸泡目标不会干出在试验过程中的时间。 根据显微镜可能会对保持在f的问题OCUS在最初的实验时间。因此，最好离开芯片为一到两小时，在显微镜载物台上装载细胞，在实验开始之前，因此它可以安定下来。 4。酵母细胞加载到微流控芯片<ol><li>连接鲁尔尖端的5毫升注射器20号鲁尔存根和一个粗管（ 约 3厘米）。 </li><li>以被加载到芯片的细胞培养物的样品。 </li></ol>用于加载的首选细胞计数是1-5×10 6个细胞每毫升之间。细胞计数较高的文化与装货前需要稀释。 YPD液体培养基上的培养细胞时，细胞装载效率可以提高许多倍，如果第一洗涤细胞并重新悬浮在基本培养基中含有葡萄糖类似的百分比在加载之前。在操作过程中的芯片，YPD培养基中可以再次被安全地使用。 <ol start="3"><li>约1毫升的细胞悬浮液装入到第ê注射器和删除所有的空气从注射器。 </li><li>注射泵的流率减少到0.5微升/分钟。在加载过程中的注射泵（步骤4.5）中所执行的低和续流将推非贴壁细胞，通过侧流路（ 图2C）。 </li><li>将含有细胞悬浮液的注射器管的出口通道。装入注射器的柱塞上轻轻按细胞。观察细胞和稳定眼下面的micropads的通过，或通过在计算机屏幕。 </li><li>在柱塞上保持压力，直到有足够的细胞下方垫定居。最佳负载是1-3细胞每垫。随着细胞定居下方的垫片随机，它是相当常见的几个垫不包含任何细胞及其他垫完全充满细胞。 </li><li>用于细胞的加载和从薄管出口通道连接到它的厚管上取下注射器，冲洗通道的一侧annel在较高流速（10微升/分钟）为一，两分钟，以除去气泡和/或还没有退出芯片（ 图2B）的细胞。如果细胞不会被删除从侧面通道，他们可以开始增长，侧通道，并与实验后的干扰。 </li><li>将回落到0.5微升/分钟的注射器泵的流量，并通过将其连接到厚的管（ 约 5厘米长），在其端部含有一个导管插件（ 图2D），关闭侧通道。 </li><li>增加至终1-5微升/分钟的流速流动。流速可能取决于介质和酵母菌株。 </li><li>启动一个显微镜和相机设置适合为目标的实验电影。确保显微镜载物台的速度被设置为油可以按照这样的低的速度。对于复制寿命，图像细胞每10分钟测定。保护细胞免受微量的卤素所发出的UV光灯，DIC采集过程中使用的，最好是在光路中放置一个附加的紫外线过滤器。使用荧光显微镜观察时，它是优选的图像较少的细胞（ 例如每20分钟），以避免光毒效应。实验完成时，在实验开始时的所有加载的细胞是死的，在正常条件下是需要长达5天。建议您定期检查的重点在实验过程中的图像，并在必要时调整。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

这里所描述的微流体方法是一个重要的新工具，在老龄化的研究，因为它使酵母复制寿命数据相结合的简单和自动化生成连续的高分辨率成像。这些属性是经典的夹层法在实验的可能性的重大改进，但需要加以考虑的方法，该方法也有一些局限性。 请注意，所确定的复制寿命可受保留母细胞:下micropad保存的每一个细胞的效率有一定概率被冲刷掉从芯片（ 例如相邻小区的出芽细胞可以把它相差的micropad）​​。集成的一个单元格的概率被冲刷掉的增加而增加的寿命。因此，它更可能是短命的细胞比长寿命的细胞被冲走之前，完成其生命周期。事实上，类似的立<html>显微切割方法与经典11-13生成的微流控设置4 fespan数据表明，这个潜在的问题是只有轻微的，即使不是无足轻重。 重要的是要注意，与微流体设置选择原始的子细胞，在实验开始时，在经典的夹层法是不可能的。然而，当加载到芯片从成倍增长的液体培养细胞，加载的细胞多数刚出生的或相对年轻（ 即约54％的细胞永远不会发芽前和一次27％左右）14。这意味着，寿命1〜2代最低估。在例外情况下，显着改变的细胞群的年龄结构，它是未能丰富处女女儿在装载前的细胞群， 例如 ，通过梯度离心分离或淘析。 ntent&gt;的微流体芯片，原则上可以被用来在不同的培养基中，研究单倍体酵母菌株。但是，不同的酵母菌株和/或介质，可能需要一些优化的流率，以便有效地保留母细胞，当细胞被保留根据它们的大小，这是不可能的加载，显着大于或小于3-4微米的直径，如二倍体酵母细胞的酵母菌株。然而，生产具有不同的光致抗蚀剂的晶片，微流体芯片可以具有不同的间距产生之间玻璃盖和micropads的允许加载这些不同尺寸的细胞。 虽然这里介绍的协议描述了如何使一个芯片上与一个单一的通道，它有可能增加在单一芯片上的多个通道并同时平行实验。根据所需的通道的数目在单个芯片中，可能有必要创建一个适合的晶片在该通道的间距更紧密地联系在一起。

芽殖酵母是一种重要的模式生物衰老研究1。直到最近，研究在酵母细胞中复制老龄化是一个艰苦的过程，需要解剖的方法，其中每个芽母细胞2,3手动删除。为了解决这个问题，我们最近提出了一种新的微流体设置能够跟踪个人母细胞在其整个寿命4。 在我们的微流控芯片，酵母细胞被困在软弹性micropads（ 见图1）。的连续流动的介质冲走新形成的女儿细胞，并提供新鲜的养分的细胞。在一次实验中，高达70母细胞可以监测在一个完全自动化的方式在其整个的复制寿命。由于微流体芯片的优异的光学性能，它可以同时监测不同方面的酵母细胞生物学（ 例如 </em&gt;的使用荧光蛋白）。 古典夹层法相比，微流控的设置提供了巨大的优势。它确保了明确和稳定的环境下进行在整个老化实验。它不需要昂贵的专用设备，并可以运行在任何显微镜配备自动对焦和时间推移能力以及温度控制细胞培养。微流控芯片的生产和经营的，可以学到在几天之内。此外，细胞可以被直接加载从指数生长培养，另一个最近发表的微流控方法5，这需要母细胞的生物素化的优点在于比高分辨率成像，在这里描述的方法可以用来测量逐步改变一个结合在酵母细胞形态，蛋白质表达和定位以前所未有的方式老化。能力长期监测单细胞酵母细胞周期的研究也提供了独特的可能性。 最近一个这种方法已经被优化，去除生物素的协议，这是16时发布的这份手稿是在审查。

<table border="1">
 <tbody>
 <tr>
 <td>Name </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>DC Sylgard 184 elastomer </td>
 <td>Mavom bv </td>
 <td>1060040 </td>
 <td>This package contains PDMS base and PDMS curing agent.</td>
 </tr>
 <tr>
 <td>Glass Petri dishes 120/20 mm </td>
 <td>VWR International </td>
 <td>391-2850 </td>
 <td></td>
 </tr>
 <tr>
 <td>Cover glasses 22x40 mm </td>
 <td>CBN Labsuppliers BV </td>
 <td>190002240 </td>
 <td></td>
 </tr>
 <tr>
 <td>Tough-Tags </td>
 <td>Sigma-Aldrich </td>
 <td>Z359106 </td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum foil </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic disposable cup </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Serological pipette 5 ml </td>
 <td>VWR International </td>
 <td>612-1245 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scotch tape </td>
 <td>VWR International </td>
 <td>819-1460 </td>
 <td></td>
 </tr>
 <tr>
 <td>Baysilone paste (GE Bayer silicones) </td>
 <td>Sigma-Aldrich </td>
 <td>85403-1EA </td>
 <td></td>
 </tr>
 <tr>
 <td>PTFE microbore tubing, 0.012"ID x 0.030"OD </td>
 <td>Cole Parmer </td>
 <td>EW-06417-11 </td>
 <td>Referred to as thin tubing </td>
 </tr>
 <tr>
 <td>Tygon microbore Tubing, 0.030"ID x 0.090"OD </td>
 <td>Cole Parmer </td>
 <td>EW-06418-03 </td>
 <td>Referred to as thick tubing </td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>VWR International </td>
 <td>233-5334 </td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml Luer-Lok syringes </td>
 <td>BD </td>
 <td>300137 </td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringes, Luer tip </td>
 <td>VWR International </td>
 <td>613-1599 </td>
 <td></td>
 </tr>
 <tr>
 <td>Tweezers </td>
 <td>VWR International </td>
 <td>232-2132 </td>
 <td></td>
 </tr>
 <tr>
 <td>20 Gauge Luer stubs </td>
 <td>Instech Solomon </td>
 <td>LS20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filters (pore size 0.20 &mu;m) </td>
 <td>Sigma-Aldrich </td>
 <td>16534K </td>
 <td></td>
 </tr>
 <tr>
 <td>Stainless steel catheter Plug, 20 ga x12 mm </td>
 <td>Instech Solomon </td>
 <td>SP20/12 </td>
 <td></td>
 </tr>
 <tr>
 <td>Petri dishes </td>
 <td>VWR International </td>
 <td>391-0892 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> EQUIPMENT</td>
 </tr>
 <tr>
 <td>Benchtop UV-Ozone Cleaner </td>
 <td>NOVA Scan </td>
 <td>PSD-UVT </td>
 <td></td>
 </tr>
 <tr>
 <td>Harvard Pump 11 Elite </td>
 <td>Harvard Apparatus </td>
 <td>70-4505 </td>
 <td></td>
 </tr>
 <tr>
 <td>SU-8 silicon master mold (wafer) </td>
 <td></td>
 <td></td>
 <td>Self-made; For details contact corresponding author </td>
 </tr>
 <tr>
 <td>Balance </td>
 <td>Sartorius corporation </td>
 <td>ED4202S </td>
 <td></td>
 </tr>
 <tr>
 <td>Vacuum pump </td>
 <td>KNF Neuberger </td>
 <td>N022 AN.18 </td>
 <td></td>
 </tr>
 <tr>
 <td>Desiccator </td>
 <td>VWR International </td>
 <td>467-2115 </td>
 <td></td>
 </tr>
 <tr>
 <td>Hot plate </td>
 <td>VWR International </td>
 <td>460-3267 </td>
 <td></td>
 </tr>
 <tr>
 <td>Optional: Metal holder for cover glass (22x40 mm) </td>
 <td></td>
 <td></td>
 <td>Self-made; For details contact corresponding author </td>
 </tr>
 <tr>
 <td>(Fluorescence) Microscope with 60x objective, autofocus, time-lapse abilities and preferably an automated (motorized XY control) stage </td>
 <td>Nikon </td>
 <td>Eclipse Ti-E </td>
 <td></td>
 </tr>
 </tbody>
</table>

continuous high-resolution microscopic observation of replicative aging in budding yeast

我们展示了使用一个简单的微流控设置，单芽殖酵母细胞可以在其整个生命周期跟踪。利用微流控芯片使用数组micropads的母亲和女儿细胞之间的大小差异。在加载时，细胞下面这些micropads，被困的的micropad和盖玻璃之间的距离，因为类似的酵母细胞（3-4微米）的直径。的加载过程后，培养液中连续冲洗通过芯片，它不仅在整个实验中，创建一个常数和定义的环境，但也刷新新兴的子细胞，后者由于其更小的尺寸下面的焊盘不保留。设置如此有效地保留了母细胞，在一次实验中，最多50个单个单元格可以监测在一个完全自动化的方式，连续5天，或者，如有必要，更长。此外，该芯片的优异的光学性能允许高高分辨率成像在整个老化过程的细胞。

在这个协议中，细胞被装入微流控芯片，直接从指数中的文化。被困在微流控芯片的细胞的年龄分布，以确定是否是类似文化装货前，细胞染色小麦凝集素结合FITC（WGA-FITC）可视化芽痕。在图3中可以看出，细胞包封微流体芯片micropads下不偏细胞的目标年龄。 自我复制的寿命可以简单地确定计数芽是由一个单一的母细胞的数量。此数据被变换成的寿命曲线绘制的反对数​​产生的芽的存活细胞的百分比。统计测试，如那些由Kaeberlein， 等。9，可以用来比较的寿命数据，从不同的实验， 图4示出一个例子的寿命曲线获得一WT BY4741酵母菌株和两个突变体（SIR2Δ，Δfob1）。 电影1时间推移电影单一的WT BY4741 YPD培养基上生长的酵母细胞。图片每10分钟。细胞产生一共有30个芽，它死之前。比例尺，5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50143/50143movie1.avi" target="_blank">点击这里观看电影</a> 。 微流控方法的一个重要优点是能够使用连续高分辨率成像。监测线粒体的形态变化，细胞老化，老化实验进行BY4742酵母细胞表达-GFP的ILV3，这是针对线粒体。 图4给出了一个概述线粒体形态的细胞为同一组在复制不同年龄（ 0芽，10芽和前死亡）。中年细胞的形态变化类似于报道文学前10名。虽然在此特定的代表性的实验中，介质的流速不是最优的，保留的子细胞具有相对高的频率对比电影1，实验的质量仍然是足以在他们的分析在49个个体的细胞的线粒体的形态整个生命周期。 电影2时间推移电影的单BY4742酵母细胞中表达，这是用于可视化的线粒体ILV3-GFP。图片每30分钟。明场以及GFP图像背景校正前两个通道合并成一个单一的图像使用ImageJ。比例尺，5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50143/50143movie2.avi" target="_blank">点击这里观看电影</a> 。 <img alt="图1" src="/files/ftp_upload/50143/50143fig1.jpg" /> 无花果余吕杏茜1。微流控的设置的示意性概览。的PDMS micropad和护罩玻璃之间的高度差是类似的酵母细胞的直径（约3-4微米）。装载:酵母细胞加载下P​​DMS micropads。培养:新鲜的介质流向连续被困细胞（蓝色箭头）。夹层:新兴子细胞冲走的流动介质（橙色箭头）。 <img alt="图2" src="/files/ftp_upload/50143/50143fig2.jpg" /> 图2。微流控芯片的通道设计。示意图。黑圆圈表示的冲孔的位置的上面的端部的入口，出口和侧流路B。介质流过的进口通道（10微升/分钟）通过较大的侧信道（橙色箭头）并退出。有没有出口通道流过，因为t之间的电阻差他侧和出口通道。细胞被加载到芯片（黑色箭头）通过出口通道。透过侧流路的流量降低到0.5微升/分钟，D:，冲洗侧流路后，侧通道被阻塞，介质中开始流过的细胞被困下面的micropads的（1-5微升/分钟）。 <img alt="图3" src="/files/ftp_upload/50143/50143fig3.jpg" /> 图3。电池装上芯片不偏向某个细胞的年龄。中旬指数YSBN6的WT文化（10 7细胞/ ml）WGA-FITC（2微升5毫克/毫升原液每10 6个细胞）标记1.5小时。细胞，然后放在载玻片上或装入微流控芯片。 Z图库（15 z轴切片在0.8微米的距离），在使用63X倍率水浸物镜的激发波长为470nm。每个芽痕的数量细胞人手点算。芽痕，决定从75个细胞的文化和79细胞的微流控芯片。 <img alt="图4" src="/files/ftp_upload/50143/50143fig4.jpg" /> 图4。获得BY4741 WT（N = 76），SIR2Δ（N = 63）和fob1Δ组（n = 58），使用这里描述的微流体装置的寿命曲线的例子缺失有一个强大的，但相反的，对测量的影响中复制的酵母细胞的寿命。每个实验中，将菌株在YPD培养基中生长过夜固定相，然后使之恢复指数生长稀释到新鲜的YPD培养基中，孵育在30℃下为3小时，在实验开始前。 Lee 等人的方法，由数据。 <img alt="图5" src="/files/ftp_upload/50143/50143fig5.jpg" /> 图5。线粒体的形态作为年龄的函数。（白色箭头）在单个细胞中线粒体的形态与年龄相关的变化:一个典型的例子。所有图像都相同的缩放。比例尺，5微米B:在一组49细胞线粒体形态观察后，他们的第一个芽（0）10的味蕾（10）和死亡（†）前。每个形态类的一个示例图像。提出的数据，得到一个单一的实验过程中，一组相同的细胞，用于在每个时间点的得分形态。线粒体可视化在BY4742菌株表达GFP标记的版本的线粒体蛋白ILV3的（来自GFP收集数据库15获得）。明场以及GFP图像背景校正前两个通道合并成一个单一的图像使用ImageJ。

Watch this Scientific Journal Video about Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast at JoVE.com

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

在这里，我们描述了一个完整的复制和/或按时间顺序寿命期间的单芽殖酵母细胞允许连续和高分辨率的显微成像的微流体装置的操作。

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

continuous-high-resolution-microscopic-observation-replicative-aging

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.8K 次观看.  University of Groningen. 在这里，我们描述了一个完整的复制和/或按时间顺序寿命期间的单芽殖酵母细胞允许连续和高分辨率的显微成像的微流体装置的操作。

视频: Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

采用两种 Electrohydrodynamic 射流的高分辨率模式: 需求下降和近场静电纺丝

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

使用 pDawn-Ag43 的高分辨率图案生物膜沉积

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...