我们承认，从教授朱迪思阿米蒂奇（英国牛津大学）和康拉德Mullineaux教授（英国伦敦大学玛丽皇后学院）组菌株的实物捐赠。 IMD是共同出资的生物化学部（牛津大学）和OCISB; AR是由工程和物理科学研究理事会（EPSRC）的DTC的助学金的资助; ND是从生物技术和生物科学研究理事会（BBSRC）资助; MCL由英国皇家学会大学研究奖学金。

<ol><li>要开始此过程中，50μL荧光蛋白表达大肠杆菌细菌细胞冷冻库存首次解冻和成长翻天覆地的5毫升LB生长介质一夜之间在37度C.在早上有氧，提取50μL这个饱和文化和子成最小的M63葡萄糖培养基，在30度4至6小时的彗星孵化，培养。在这里，我们展示了使用两种不同的细胞株，其中一个表示电子传输的细胞色素融合到绿色荧光蛋白，表示有关的蛋白质融合，以绿色荧光蛋白在细菌鞭毛电机的其他。 </li><li>细胞可收获直接从不断增长的亚文化，如果他们作为固定样本观看，或他们可能会剪截断细菌鞭毛“拴”条件下观看。 </li><li>剪切涉及配售的亚文化通常为1 -5毫升成两个无菌注射器，无菌管道加入的设备。剪切是通过在每个注射泵交替推推动通过狭窄的管文化。这是50-100倍，所需的剪切程度而定。文化是离心沉淀细胞，它是悬浮在最短的媒体删除鞭毛片段。 </li><li>然后，我们准备在氢​​氧化钾饱和溶液浸泡20分钟乙醇清洗BK7玻璃盖玻片，然后在去离子水和乙醇和漂洗彻底离开干燥的空气中至少有1小时</li><li>我们构造一个简单的流细胞内部的细胞，在显微镜。这涉及到一个BK7玻璃显微镜幻灯片上绘制石蜡油脂线，然后建立一个隧道，夹层置于顶部的清洗coverlips之一，并与一双镊子轻轻按下，让流细胞体积的5-10μL 。 </li><li>观察固定化细胞注射填充流动细胞与聚- L -赖氨酸0.1％W / V解决方案，并允许它在室温下孵育至少1分钟。然后，我们冲洗出来使用100μL最小的媒体，通过注入流通池的一端从其他媒体和排汗用纸巾。 </li><li>然后，我们通过一个直径为200纳米乳胶微球（Polysciences）1:500稀释在最短的媒体，以纪念盖玻片表面的20μL灯芯。流通池倒盖玻片朝下，放置在一个平台上清晰的表面，在一个简单的湿度室，并在室温下孵育5 min。未绑定的珠子，然后通过100μL最小媒体排汗冲走。 </li><li>如果我们想观察拴细胞，我们省略了聚- L -赖氨酸的一步，而不是填补流动细胞与5 mg / ml的抗鞭毛蛋白抗体。流通池是在10分钟的湿度室的地方，然后通过排汗刷新。 </li><li> 20细胞培养液，然后恶人通过流通池，可以使用剪切样品，如果观察拴细胞固定化细胞在观看或unsheared样品。 </li><li>倒流通池，并放置在湿度为20分钟室。未绑定的细胞，然后通过100μL最小媒体排汗冲了出去。 </li><li>浸油下降是摆在盖玻片的顶面中心和流通池，然后轻轻地放在定制荧光显微镜的样品架，使高数值孔径物镜的光学接触。 </li><li>显微镜电子倍增接通相机和相机设置被冷却到-70摄氏度，该软件是掌握在一个典型的帧速率，帧传输模式，在25赫兹的图像，最初disenabling上的增益控制摄像头。 </li><li>明照明开启和图像带入重点，选择一个合适的细胞或细胞组，他们被卡，其​​长轴平行的盖玻片表面的基础上成像。重点是微调，以确保200纳米乳胶珠坚持盖玻片表面，只是在重点。 </li><li>图像序列是在明收购记录的细胞体的轮廓。明照明关闭和启用摄像机的增益为最大。 </li><li>对于收购标准，使用全内反射荧光（TIRF）通过适当波长的激光束（在这里，绿色荧光蛋白的激发，我们使用了473纳米激光）是预先设定，以便集中后焦平面客观的镜头，但流离失所光轴横向到细胞样品的荧光激发产生渐逝场。 </li><li>相机采集开始，和激光快门打开激发荧光蛋白的细菌内。收购的激光强度和速度的参数需要尝试不同的价值观，特别是在调查的生物系统进行了优化，而是一个典型的范围relevant研究细胞膜的移动蛋白复合物的1-10毫瓦激光功率超过一个直径1〜30微米的圆形激发面积，每帧5-40毫秒的曝光时间。样品被点亮，直到光漂白，通常为10秒</li><li>都拴细胞，在细胞体旋转的附着点之间的盖玻片和鞭毛存根和固定化细胞固定盖玻片表面，可以使用此协议。 </li><li>有一个复合物的高拷贝数的一些细胞株细胞TIRF成像之前的一极的重点从最初的衍射极限的激光漂白受益。这种漂白后荧光恢复使用漂白（FRAP），相当于。使用我们的自定义显微镜激发激光灯可喂到第二个独立使用的路径为FRAP型漂白。通常情况下使用1 - 10mW的激光功率，一个典型的漂白时间范围在10-300毫秒。这样的结果要高得多，在允许个别的复合物，随后进入该地区扩散到更容易地可视化细胞漂白区造影。 </li><li>复合物在细胞浆内扩散速度远远比在细胞膜上采用不同的照明模式，所谓的“slimfield”的可视化。这里聚焦的激光是横向扩展，只包括一个单细胞。这将产生一个非常激烈的领域，允许采取风险通常毫秒快得多。 </li><li>经过数据采集，图像送入自定义编写的软件（在LabVIEW 8.5中的编码）。这会自动检测在一个典型的几个纳米的精度细胞的荧光斑点的位置，并提取它们的大小和亮度。 brightnessof漂白跟踪跟踪的分子复合物的时间是用来估计化学计量的，即弥补了单分子复杂多少个人的荧光蛋白质。 </li></ol>代表性的成果： 当该协议是正确的细胞在明看的图像是非常明显的，对一个白色/灰色的细胞体（图1A）黑暗的细胞体的周长。在荧光采用固定化细胞，我们可以看到踩点的强度，一般宽度在250-300纳米（图1b）。将被视为健康，拴细胞绕明图像点的系绳附件。在我们的例子中的一些分子络合物在荧光激发下也可能看到的附着点，说明标签蛋白与鞭毛马达的本地化。这些斑点是个别分子络合物，他们看到的数量将取决于所使用的照明模式，以及如何配合物的许多实际上存在于细胞内，在任何一个时间。点的流动性取决于下的具体研究生物系统。如果斑点的密度最初非常高，因为这里使用的标记细胞色素的情况下，然后执行初始FRAP还原漂白剂可以提高成像的对比度。 <img src="/files/ftp_upload/1508/1508Figure1tmb.jpg" border="0" alt="图1" /> 图1。 （一）明场和（二）TIRF图像（假色）为一个固定的大肠杆菌细胞表达的一种蛋白质融合，这是众所周知的细菌鞭毛马达的绿色荧光蛋白（GFP ）的。请<a href="https://www.jove.com/files/ftp_upload/1508/1508Figure1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

The authors have nothing to disclose.

必须小心不要“剪切”拴细菌细胞，因为这可能会损害鞭毛马达的功能。重要的是使用细胞的显微镜玻片上一次更长的时间不到一个小时，因为他们可能成为氧气耗尽。可能需要相当大的优化，正在调查中找到最好的显微镜成像条件满足您特定的生物系统。这可能是明智的做法是企图用纯化GFP的单独确定正确的强度激光激发您的特殊的显微镜系统所需的成像。

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

全面洞察到活细胞的机制，可以实现只有通过调查的关键工序引出并直接在细胞水平上的事件。迄今为止的生物系统的剪切复杂性造成了精确的单分子实验过于苛刻，而不是单一系统使用相对粗糙的散装合奏平均测量的研究重点。然而，许多重要的过程发生在活细胞只是一个水平或几个分子;合奏测量一般掩盖这些事件的随机性和异质性。在这里，使用先进的光学显微镜和分析图像分析工具，我们演示了如何在一个单一的生活细菌细胞的单个分子的精确度，以及如何运作的生物机器，我们可以观察到分子复合物内的动态监测的蛋白质。该技术直接相关的生理。他们微创微扰和非侵入性正在研究的生物样品，并完全合拍，在物质生活的调查功能，没有现成的其他单分子生物物理的方法。此外，生物标本研究所有在未修改的细胞株（“基因组编码”），产生显着更多的蛋白质比自然发生的，而不是比较常见的，但不太理想的方法几乎相同的水平产生荧光标记的蛋白质（“质粒表达”）。因此，将调查的实际生物样品中有显着接近自然的有机体，因此观测真正的生理过程有关。

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

在这里，我们展示了单分子荧光显微镜，对生活的细菌细胞，使功能分子复合物进行检测，跟踪和量化的执行协议。

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

visualizing-single-molecular-complexes-vivo-using-advanced

Research

JoVE Journal

Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

9.2K 次观看.  University of Oxford. 在这里，我们展示了单分子荧光显微镜，对生活的细菌细胞，使功能分子复合物进行检测，跟踪和量化的执行协议。

视频: Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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 ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...