我们感谢他的帮助在开发系统的微流体装置的设计和设置加工零件在哈佛大学罗兰研究所的工作人员的意见，约书亚埃德尔德兰Sabanayagam。我们承认了有益的讨论，与梅勒的研究小组的成员罗兰研究所和波士顿大学，并在伊利诺伊大学的T.房委会“组的成员有用的信息。最后，我们对此表示赞赏从国家科学基金会（PHY - 0701207）和美国国立卫生研究所（GM075893）的财政支持。

1 - 组装微流体细胞微流体细胞是通过融合作出了图案，2毫米厚的聚二甲基硅氧烷（PDMS）磁盘新鲜清洁的石英盖玻片。 PDMS的磁盘包含四个30μL，入口和灵活的毛细血管连接到一个缓冲区水库和注射泵，分别为控制流量的缓冲区，出口访问的矩形商会。支持流通池玻璃窗的背面，并放在一个特制的细胞含有水冷却热电元素来调节温度的持有人。 <ol><li>为了使图案的PDMS磁盘，10个部分的有机硅弹性体基地与第1部分的固化剂混合，拌匀，并离开1小时在真空脱气。 </li><li>轻轻倒入到微细胞模具的弹性体组合。在我们的例子中，这是1负型光刻胶的4条（1 × 4 × 1 × 3 × 300微米）的硅晶片。离开这个治愈一个热点板块在70 ° C为两个小时。 </li><li>小心剥离固化后的PDMS使用刀片模具磁盘。融合到一个1英寸的玻璃窗口等离子的氧化两面为30秒（含8 x 2毫米直径预钻孔在通道的两端）的磁盘。保险丝上的PDMS盘平面一侧的车窗玻璃（不包含渠道方）。 </li><li>通过每一个玻璃窗口的孔中插入一个2英寸长的灵活熔融石英毛细管，直到它到达通道。先插入一根针，然后下面的毛细血管可能促进这一进程。使用快速固化硅铸造佳铸造等复合密封玻璃窗孔。 </li><li>用乙醇和水冲洗渠道，血浆与新鲜清洁，1英寸的圆形为30秒石英盖玻片激活细胞一起。我们建议清洁盖玻片使用食人鱼*.保险丝的盖玻片一侧的细胞含有渠道，密封的商会。 </li><li>离开组装的电池在室温下固化过夜。 </li></ol> *食人鱼是解决方案的H 2 O 2和H 2 SO 4 1:2.5的比例。要干净盖玻片，沉浸在食人鱼在90 ° C为20分钟。 2 - 激活分子固定的表面核酸研究的，简单的表面固定计划由第一层玻璃或石英盖玻片与生物素标记的牛血清白蛋白（BSA）与链霉亲。这允许被固定在室温天高特异性生物素标记的样本。 <ol><li>软管连接，方便的缓冲区，使用注射器和针头注射的毛细血管。 </li><li>注入0.1毫克/毫升的缓冲液中的生物素化的牛血清白蛋白溶液（10毫米的Tris - HCl，pH值8.0，50 mM氯化钠，使用0.02微米的过滤器过滤）进入通道和至少30分钟的孵育。 </li><li>一个流量300-500缓冲液从通道中删除的任何生物素标记的牛血清白蛋白，不小心陷阱箱内任何气泡。 </li><li>注入溶液的0.1毫克/毫升缓冲液和素A和孵育15分钟。然后重复步骤3。 </li><li>流通过一个生物素标记的样本下午3:00-5:00解决方案。执行表面扫描检查的荧光分子的覆盖面。如果有必要，增加样品的浓度，以取得更好的覆盖。孵育10分钟，并重复步骤3。 </li></ol>微流体细胞是安装在机动XY允许粗（高达25毫米）的议案采用直流马达光学编码阶段，并使用两个闭环压电执行器的罚款（1纳米）的议案（Physik公司Instrumente模型M - 014或类似的）。这可以激活表面样品固定之前或之后。 3 - 准备成像缓冲器（IB） IB由一种酶氧清除系统和三重淬灭，如Trolox的。由于IB是通过不断一夜之间刷新，至少有10毫升，应提前做好准备。 <ol><li>准备10毫升0.8％W / V D -葡萄糖，至少有35毫米的Tris - HCl pH值8.0，氯化钠和50毫米，在去离子水。较高浓度的缓冲重要的是有两个原因：溶解Trolox的酸化的解决方案，并也将降低酶反应随着时间的推移溶液的pH值。由于该解决方案具有保质期长，它可以存储作为原液。 </li><li>溶解在5毫克Trolox的振荡几分钟，然后摇晃10分钟，在第1步准备的缓冲区。 Trolox的不是很易溶于水，在pH&gt; 7，所以不要急于这一步。过滤的解决方案，使用一个0.2微米的过滤器，并保持在10分钟的真空脱气。 </li><li>准备通过混合60μL瓦特的“gloxy”酶的解决方案R，20μL5X缓冲，过氧化氢酶20μL，1毫克的葡萄糖氧化酶和100μL10 mg / ml的BSA。使用0.22微米的离心过滤器过滤的解决方案。 </li><li> “gloxy”解决方案，避免形成气泡从步骤3，轻轻地从第2步混合缓冲区。 </li><li>使用机械注射泵控制流速在5μL/ min的恒定速率的通道流过的IB。泡泡氩气进入IB水库重新溶解到缓冲区，以防止大气中的氧气。 </li></ol>表面扫描，分子本地化和收购单分子的强度痕迹，是控制一个LabVIEW程序，允许自动数据收集 1 。该计划在0.2μm的分辨率扫描20X20微米2地区，0.1微米/ MS率。数据立即处理，屈服强度加权上述背景计数的所有像素的位置。固定分子一旦被发现，他们都感动到焦的供体和受体荧光基团的数量和强度是记录的时间，直到两个荧光基团的光漂白的功能。编程阶段是移动到一个新的原产地100微米，重复扫描过程。 4 - 代表性的结果下面是一些组装的微流体细胞表面活化前和安装后，正准备分子固定在显微镜图像显示。如果通道被激活成功，表面扫描应类似于下面显示后直接捐助激发捐助染料（绿色）和受体（红色）的排放所描绘的扫描。这些分子移动一进入探测量，超过时间，直到两个染​​料的光漂白和荧光记录，在单个分子的痕迹。来自其中之一的痕迹，可以得到的FRET效率，瞬时体 - 受体的分离，这又是用来探索生物的兴趣分子的结构和动态通知。 <img src="/files/ftp_upload/1542/1542fig1tmb.jpg" alt="图1" border="0" /> 图1：四通道微流控细胞，每个入口/出口的毛细血管。请<a href="https://www.jove.com/files/ftp_upload/1542/1542fig1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1542/1542fig2tmb.jpg" alt="图2" border="0" /> 图2：安装在显微镜测量准备的细胞。请<a href="https://www.jove.com/files/ftp_upload/1542/1542fig2.jpg">点击这里</a>看大图图2。 <img src="/files/ftp_upload/1542/1542fig3tmb.jpg" alt="图3" border="0" /> 图3：SM - FRET技术测量的设置。图3看到一个更大的版本， <a href="https://www.jove.com/files/ftp_upload/1542/1542fig3.jpg">请点击</a>这里。 <img src="/files/ftp_upload/1542/1542fig4tmb.jpg" alt="图4" border="0" /><img src="/files/ftp_upload/1542/1542fig5tmb.jpg" alt="图5" border="0" /> 图4和图5：红色和绿色通道的一个固定的表面扫描FRET绿色激光激发的分子。请点击查看大图<a href="https://www.jove.com/files/ftp_upload/1542/1542fig4.jpg">图4</a> ， <a href="https://www.jove.com/files/ftp_upload/1542/1542fig5.jpg">图5</a> 。 <img src="/files/ftp_upload/1542/1542fig6tmb.jpg" alt="图6" border="0" /><img src="/files/ftp_upload/1542/1542fig7tmb.jpg" alt="图7" border="0" /> <img src="/files/ftp_upload/1542/1542fig8tmb.jpg" alt="图8" border="0" /><img src="/files/ftp_upload/1542/1542fig9tmb.jpg" alt="图9" border="0" /> 图6，7，8，9：强度轨迹的捐助（绿色）和受体（红色）标记的一个DNA分子8，10，16和18个碱基对，除了的荧光基团。请点击看到一个更大的版本<a href="https://www.jove.com/files/ftp_upload/1542/1542fig6.jpg">，图</a> 6 <a href="https://www.jove.com/files/ftp_upload/1542/1542fig7.jpg">，图</a> 7 <a href="https://www.jove.com/files/ftp_upload/1542/1542fig8.jpg">，图</a> 8 <a href="https://www.jove.com/files/ftp_upload/1542/1542fig9.jpg">，图</a> 9 。

<ol>
	<li>Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Techniques,+a+Laboratory+Manual.">Single-Molecule Techniques, a Laboratory Manual.</a> CSHL Press. , (2008).</li><li>Sabanayagam, C. R., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+scanning+confocal+microscope+for+single+molecule+analysis.">High-throughput scanning confocal microscope for single molecule analysis.</a> Applied Pys. Letters. , 84-87 (2004).</li><li>Sabanayagam, C. R., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Fluorescence+resonance+energy+transfer+to+measure+distances+along+individual+DNA+molecules:+Corrections+due+to+nonideal+transfer.">Using Fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer.</a> J. Chem. Phys. 122, 61103-61105 (2004).</li><li>Echeverra, A., Aitken, C., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Oxygen+Scavenging+System+for+Improvement+of+Dye+Stability+in+Single-Molecule+Fluorescence+Experiments.">An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments.</a> Biophysical Journal. 94, 1826-1835 (2008).</li><li>Rasnik, I., McKinney, S. A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonblinking+and+long-lasting+single+molecule+fluorescence+imaging.">Nonblinking and long-lasting single molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li><li>Boukobza, E., Sonnenfeld, A., Haran, G., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+in+Surface-Tethered+Lipid+Vesicles+as+a+New+Tool+for+Single+Biomolecule+Spectroscopy.">Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy.</a> J. Phys. Chem. 105, 12165-12170 (2001).</li><li>Di Fiori, N., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Article in preparation. , .</li></ol>

虽然上述协议已经为我们特定的系统和优化的染料（TMR和ATTO647N）的具体选择，它决不是唯一的证明方法。近日，原儿茶酸（PCA）/ protocatechuate 3,4 -双加氧酶（PCD）组成的替代氧清除系统增加 4某些染料的耐光性。同样，其他如β-巯基乙醇（BME）的三重态的猝灭可以用来代替Trolox的，尽管这些可能无法抑制长期闪烁一些染料，特别是Cy5的5。 虽然固定通过生物 ​​素-链霉菌抗生物素标记的牛血清白蛋白（BSA）是核酸研究的首选，更适合一个聚乙二醇（PEG）涂层表面的蛋白质的研究，因为它可以防止非特异性粘附1。此外，子衍射极限的大小水泡，可用于封装生物分子在利益的案件可以由表面相互作用6的影响。 在这篇文章中，我们已经使用共聚焦设置，配备与硅雪崩光电二极管获得的荧光强度的痕迹。共有配备CCD相机内反射荧光显微镜（TIRF），该协议同样适用。不管使用的显微镜，SM - FRET技术可以告知接近埃时，供体和受体信号的正确纠正 7的空间分辨率。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fused silica capillary tubing</td>
<td>&nbsp;</td>
<td>Polymicro Technologies</td>
<td>TSP150375</td>
<td>&nbsp;</td>
<tr>
<td>Fused silica cover slip</td>
<td>&nbsp;</td>
<td>Esco Products</td>
<td>R425000</td>
<td>&nbsp;</td>
<tr>
<td>Tubing</td>
<td>&nbsp;</td>
<td>Diba Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8549</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9085</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>0021122</td>
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<td>Sylgard 184 Elastomer Kit</td>
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<td>DowCorning</td>
<td>3097366-1004</td>
<td>Silicone Elastomer Kit</td>
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<td>Trolox</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>238813</td>
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<td>Catalase</td>
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<td>Sigma</td>
<td>C3155</td>
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<td>Glucose Oxidase</td>
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<td>Sigma</td>
<td>G2133</td>
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<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
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<td>Kwik-Cast Sealant</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Kwik-Cast</td>
<td>Silicone Casting Compound</td>
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automated system for single molecule fluorescence measurements of surface-immobilized biomolecules

荧光共振能量转移（FRET）显微镜被广泛用于研究的结构和动态，如核酸和蛋白质的分子生物的兴趣。单分子FRET（SM - FRET）测量固定分子允许长期观测系统有效，直到两种染料的光漂白时间的痕迹，从而对生物分子的动力学报告，同一个毫秒时间尺度的广泛到几分钟。为了便于收购的大量统计分析的痕迹，必须是自动化的过程，应严格控制在整个测量时间（〜12小时）和样品的环境。这是通过使用自动扫描共聚焦显微镜，让成千上万的单个分子的审讯在一夜之间，一个微细胞，允许控制的缓冲区交换，有限制的氧含量和整个测量期间保持一个恒定的温度。在这里，我们展示了如何组装的微流体装置，以及如何激活其表面DNA的固定。然后，我们解释了如何准备一个缓冲区，以便最大限度的耐光性和荧光团的寿命。最后，我们所涉及的步骤在准备设置为自动获取时间分辨的单分子FRET DNA分子的痕迹。

Watch this Scientific Journal Video about Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules at JoVE.com

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

在这篇文章中，我们描述了我们如何取得的FRET从单个DNA分子固定到表面，使用自动扫描共聚焦显微镜的痕迹。

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

automated-system-for-single-molecule-fluorescence-measurements

Research

JoVE Journal

Biology

12.8K 次观看.  Boston University. 在这篇文章中，我们描述了我们如何取得的FRET从单个DNA分子固定到表面，使用自动扫描共聚焦显微镜的痕迹。

视频: Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals.

通过共聚焦显微镜重建单细胞先天荧光特征

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...