格兰特：5RO1GM086447 02。

1。样品制备<ol><li>应变JB281的单菌落接种LB培养基[BW25113 / pJB042（P 紫胶 ：的FtsZ的mEos2）]。在振动机中生长过夜，在37℃下</li><li>文化1:1,000稀释到M9 +最小的媒体M9盐，0.4％葡萄糖，2毫米硫酸镁 ，0.1毫米氯化钙 ，MEM氨基酸和维生素]和成长中旬至对数生长期（OD 600 = 0.2-0.3）氯霉素（150微克/毫升）的存在下，在室温（RT）。 </li><li>诱导文化与20μMIPTG 2小时（见注1）。 </li><li>颗粒的离心（8000转，1分钟），重悬在M9 +，重复相同体积的文化。所述第二再悬浮后，在RT下继续生长2小时。 </li><li>修复诱导培养，用4％多聚甲醛的PBS（pH值= 7.4），在RT下40分钟。团培养等体积的PBS重悬，重复。如果活细胞成像，与等量跳过固定，球团文化，重悬的M9 - [M9的盐，0.4％葡萄糖，2mM的用MgSO 4，0.1mM的CaCl 2的 [MEM氨基酸;重复。 </li><li>稀释黄金珠，1:10再悬浮文化。应用样品制备成像室（见2.4）。 </li></ol> [1]注：以上感应协议（步骤1.1-1.3）表达系统的应变JB281进行了优化。详细的诱导条件下可能会有所不同其他系统或蛋白质的表达。 2。大会影像厅<ol><li> 3％（重量/体积）的琼脂糖溶液在M9 - 。熔化琼脂糖在70℃下在台式热块为40min。专区熔化琼脂糖在50℃达5小时。 </li><li>将一个洁净的microaqueduct幻灯片中的上半部分与电极面朝下的摄像室。对齐的下垫圈上microaqueduct幻灯片，以便覆盖灌注通道。 </li><li>应用〜50μl的熔化的琼脂糖的中心的玻璃幻灯片。立即琼脂糖凝胶滴用干净，干燥的盖玻片。使凝胶在室温固化30分钟。 <ol><li>要清洁盖玻片：安排在一个陶瓷支架的盖玻片，超声处理20分钟，在旋转浴，丙酮，乙醇和1M KOH在玻璃罐。重复循环三次，共9超声处理，然后由一个单一的超声处理在蒸馏水中。将清洗盖玻片新鲜蒸馏水中。使用前，将过滤后的空气吹干盖玻片。 </li></ol></li><li>小心地取出盖玻片，使凝胶垫的microaqueduct的幻灯片。立即加入1微升的细胞培养样品（见步骤1.6）的凝胶垫的顶部。等待〜2分钟，使溶液被吸收的凝胶垫。顶部硅胶垫一个新的干净，干燥的盖玻片。装配整体成像室根据制造商的说明。 </li></ol> 3。图像采集<ol><li>打开的显微镜，照相机和激光器。打开的MetaMorph软软件（Molecular Devices公司）。 </li><li>将适当的激发/发射滤波器的摄像路径中（ 图1）上。 </li><li>将激光功率和相应的采集设置。 <ol><li> 488纳米激光= 15毫瓦（见注2） </li><li> 561 nm激光= 75毫瓦</li><li> 405 nm激光= 2兆瓦±中性密度滤镜 （见说明3） </li></ol></li><li>锁定成像室分成的阶段，通过互补阶段适配器。 </li><li>指定一个合适的成像区域（100x100像素），均匀照亮所有三个激光器。 </li><li>确定样区，包含细胞和基准标记（黄金珠）非常接近，但不重叠。 </li><li>集中最接近的盖玻片的细胞的表面上。一个明场图像采集与整合时间为50毫秒，移动焦点到0.5微米的样品（ 图3AI）。 </li><li>合奏与激发绿色荧光图像采集的488-N<html> m激光使用的是50毫秒积分时间（ 图3Aii）。 </li><li>采集视频流在红色通道，连续照明405 nm和561 nm的激光器。对于固定的细胞中，50 ms的帧速率，总共20000张（共17分）与405纳米的激光功率斜坡〜10％，每1000帧（见注＃4）。对于活细胞中，一个10毫秒的帧速率被用于共3000帧（〜30秒总）在一个恒定的405-nm的激光功率。 </li><li>将焦点返回到的下表面上的细胞，并在步骤3.7中获得另一个明视野图像。 </li></ol> [2]注：一个细胞的绿色荧光的积分强度是成正比的FtsZ-mEos2分子在细胞中表达的总数。 488 nm激发动力和合奏荧光的采集设置应保持不变，为所有的细胞相对FtsZ的mEos2在不同细胞中的表达水平进行比较。 ontent“[3]注：固定细胞成像与中性密度（ND）滤镜（ 图1，光学组件＃5）在地方，以达到一个缓慢的启动速度，使每个单个分子可以准确识别。 ND滤光器除去成像时增加活细胞的激活率，同时保持较低的概率的两个分子，受衍射限制的区域中被同时激活。施加到活细胞成像速度是必要的，以减少总的采集时间，从而“冻结”的Z-环在时间和限制的光损伤的细胞的影响。 [4]注：收购的帧的数目进行了优化，为我们的系统中，并且依赖于特定的蜂窝结构，标签密度，活化率和，是否排出整个池荧光团是非常重要的分析。在活细胞中，重要的是在尽可能短的期间的钛，以获得足够数量的本地化我要尽可能避免蜂窝结构的移动引起的图像的模糊。 4。 PALM形象建设<ol><li>确定每个检测点的质心位置。 <ol><li>将图像堆栈到Matlab（MathWorks公司）。 </li><li>裁剪图像堆栈的区域周围一格，排除基准珠的。 </li><li>选择斑点检测，是上述的背景水平，但低于平均光斑强度的强度阈值。我们占本底水平的变化在整个实验的最大强度计算平均使用150帧窗口。然后，对每一帧的强度阈值从正在运行的平均值插值。 </li><li>减去从每一帧中的各自的阈值。准点确定为与阈值以上的强度的三个相邻像素。 </li><li>适合每个准点的荧光强度，以一个二维Gaussian函数[方程＃1]使用的非线性最小二乘算法（ 图2）。使用检测的光子的总数[等式＃2]来计算每个点的定位精度。任何不良的合适的点与定位精度大于20nm（固定的细胞）或45纳米（活细胞）（见注＃5）被丢弃。的具有的强度大于2倍的平均强度，这可能是由于重叠发射器的任何位置，也被丢弃。 </li><li>固定细胞，除去重复观察同一分子无视任何地点，其质心位置是在45纳米 任何其他点，它之前由6或更少的帧。对于活细胞，所有景点都将保留。 </li></ol></li><li>校准样品漂移基准标记的运动。 <ol><li>周围的基准标记出一个10x10像素区域的作物。 </li><li>减去在4.1.3确定的各自的阈值，从每一帧中。 </li><li>在每个适合的强度分布帧通过最小二乘法拟合算法假设一个二维高斯形状。 </li><li>计算质心的相对位置第1帧之间的位移。 </li></ol></li><li>纠正每一个独特的分子质心位置的确定在4.1与4.2计算合适的样品漂移。比较亮场图像中获得3.7和3.10。如果细胞观察到的基准标记的相对移动，数据将被抛出。 </li><li> 15×15 nm的像素组成一个单一的形象叠加校正质心位置。绘制每个唯一的分子作为一个单位区域，二维高斯分布的重心位置为中心的，其标准偏差等于定位精度[等式＃2]。这样的查询结果中的概率密度图，其中的像素的强度正比于在该象素（ 图3Aiv）找到一个分子的可能性。 </li><li>比较PALM图像到合奏图像。 <ol><li>重新绘制的中心id会在4.4的位置，但在一个平面上与150×150 nm的像素和一个标准偏差等于250nm。这会产生一个PALM模仿受衍射限制的图像，它可以被用来比较受衍射限制的，合奏中3.8（ 图3Aiii）获取的图像的图像。 </li><li>要确认所检测到的分子是代表整个人口，比较重建的合奏图像（ 图3Aiii，步骤4.5.1）与实验乐团荧光图像（ 图3Aii，步骤3.8），以确认这两个图像显示类似形状的结构，定向和相对强度。 </li></ol></li></ol> [5]注：凭经验确定所需的最小定位精度为每个成像方法对于一个给定的样本，并通过绘制所有分子的精度，选择一个合适的切断。 5。 PALM图像分析<ol><li>测量Z-环WIDTh和直径。 <ol><li>旋转对PALM图像，以便以垂直方向的长轴的细胞（ 图4A）。 </li><li>作物满分蜂窝区域含有的Z-环。 </li><li>计算累积强度投射的强度分布沿细胞的长轴。 </li><li>适合的强度分布为高斯分布。被定义为拟合的高斯分布（ 图4B）的全宽度半最大值（FWHM）的环宽度。 </li><li> 5.1.2项目的累积强度分布沿细胞的短轴。环直径的整个长度上的强度分布图（ 图4C）被定义为。 </li></ol></li><li>绘制FtsZ的密度（见注＃6）。 <ol><li>重新绘制在（4.4）中的重心的位置，但没有的高斯分布的情况下，使得每一个独特的分子被赋予一个值1，并分配给对应于它的质心位置的单个像素。在这种方式中，每个的强度像素表示该像素中检测到的分子的总数。密度PALM图像，还可以可视化的等高线图（ 图5E）。 </li></ol></li><li>确定空间分辨率。 <ol><li>从理论上讲，可以实现空间分辨率：创建一个有代表性的PSF整个样本确定的平均定位精度的检测的分子和计算FWHM（方程3）。 </li><li>实验达到的空间分辨率相同的分子之间的重复观测计算的平均位移。 </li></ol></li></ol> [6]注：我们使用全内反射的PALM（TIR-PALM， 如图1和图5），以限制上面的小区/玻璃界面的薄层（〜200 nm）的激活和激发。将被检测到，以便只FtsZ的分子与膜最接近盖玻片。

<ol>
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没有利益冲突的声明。

PALM图像包含有关分子计数和在小区内的位置的信息，允许的靶蛋白分子的分布和排列是难以通过其他方式实现的详细分析。下面，我们列出了应采取的预防措施，提取准确的定量信息，同时保持生物PALM图像的相关性。我们还探索能最好地利用活细胞与固定的信息。最后，我们建议的渠道，获得额外的超分辨率细胞分裂机械信息。 上面描述的方法进行了优化，以产生精确的PA...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 50×MEM氨基酸</td><td>西格玛</td><td> M5550 </td><td></td></tr><tr><td> 100×MEM维生素</td><td>西格玛</td><td> M6895 </td><td></td></tr><tr><td> IPTG </td><td>敏达</td><td> 46-102-RF </td><td></td></tr><tr><td> 16％的多聚甲醛</td><td>电子Micrsocopy </td><td> 15710-S </td><td></td></tr><tr><td> SeaPlaque GTG琼脂糖</td><td> Lonzo </td><td> 50111 </td><td></td></tr><tr><td> 50纳米的黄金珠</td><td>微球的纳米微球的制备</td><td> 790113-010 &lt;/ TD&gt; <td></td></tr><tr><td> FCS2成像室</td><td> Bioptechs </td><td></td><td></td></tr><tr><td>舞台适配器</td><td> ASI </td><td> I-3017 </td><td></td></tr><tr><td>倒置显微镜</td><td>奥林巴斯</td><td> IX71 </td><td></td></tr><tr><td> 1.45 NA，60倍的目标</td><td>奥林巴斯</td><td></td><td></td></tr><tr><td> IXON EMCCD相机</td><td>安道​​尔技术</td><td> DU897E </td><td></td></tr><tr><td> 488纳米蓝宝石激光</td><td>连贯</td><td></td><td></td></tr><tr><td> 561-nm的蓝宝石激光</td><td>连贯</td><td></td><td></td></tr><tr><td> 405纳米的CUBE激光</td><td>连贯</td><td></td><td></td>
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 </table>

super-resolution imaging of the bacterial division machinery

细菌细胞分裂需要协调的装配超过10种必需的蛋白质，在midcell 1,2。这个过程的核心是形成一个环形的表壳构造（Z-环）的FtsZ蛋白的划分方案3,4。的Z-环由多个单链FtsZ的原丝，并理解的Z-环内的原丝的配置将提供洞察作为力发生器5,6 Z-环组件和它的功能的机制。此信息一直难以实现，由于目前的限制，在传统的荧光显微镜和电子显微镜。无法提供一个高分辨率图像的Z-环，由于光的衍射极限（〜200 nm）的常规的荧光显微镜。电子cryotomographic成像检测散FtsZ的原丝在小C. crescentus的电池7，但很难适用于较大的细胞，例如大肠杆菌或 B。 枯草芽孢杆菌 。在这里，我们描述了一个超高分辨率荧光显微镜方法的应用，光敏定位显微镜（PALM），定量表征的组织结构的E.大肠杆菌 Z-环8。 PALM成像可以同时提供高的空间分辨率（〜35纳米）和特定的标签，使靶蛋白的明确识别。我们标记FtsZ的可光活化的荧光蛋白mEos2，切换到绿色荧光（激发波长= 488纳米）的红色荧光（激发波长= 561纳米）激活后在405 nm 9。在一个PALM试验，单的FtsZ，mEos2分子，随机激活，相应的单分子质心的位置确定精度&lt;20纳米。甲的超分辨率图像的Z-环然后重建通过叠加的质心位置的所有检测到的FtsZ-mEos2分子。 &lt;p类=的“jove_content”&gt;使用这种方法，我们发现，在Z-环具有一个固定的宽度为100 nm，并组成一个松散捆FtsZ的原丝，在三维空间中彼此重叠。这些数据提供了进一步调查的跳板的Z形环10的细胞周期依赖性变化，并可以应用到其他感兴趣的蛋白质。

在图3Aiv是示出一个两维的，超分辨率渲染从PALM的成像方法，如上所述，产生的Z-环。下面，我们总结可以由它们得到的定性和定量信息。 定性地说，我们观察到的Z形环是一个不规则的结构，该结构采用在常规的荧光图像（比较图3A-DII和iv）是无法区分的多个配置（单一条带或螺旋形的弧）。如这些观察可以被用来确定该细胞群的百分比，显示一个特...

Watch this Scientific Journal Video about Super-resolution Imaging of the Bacterial Division Machinery at JoVE.com

Super-resolution Imaging of the Bacterial Division Machinery

我们描述了超分辨率成像方法的细菌FtsZ的环探测的组织结构，细胞分裂的重要设备。这种方法是基于光活化的定位显微镜（PALM）图像的定量分析，并可以应用到其他的细菌的细胞骨架蛋白。

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

super-resolution-imaging-of-the-bacterial-division-machinery

Research

JoVE Journal

Biology

11.7K 次观看.  The Johns Hopkins University School of Medicine. 我们描述了超分辨率成像方法的细菌FtsZ的环探测的组织结构，细胞分裂的重要设备。这种方法是基于光活化的定位显微镜（PALM）图像的定量分析，并可以应用到其他的细菌的细胞骨架蛋白。

视频: Super-resolution Imaging of the Bacterial Division Machinery

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

细菌细胞的电子Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

科研

生物学

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to chromatic aberration and the imperfect alignment of cameras, images acquired in each channel may be shifted, and magnified, as well as rotated relative to each other in any of the three dimensions. With the classical calibration method, chromatic shifts are measured by multicolor beads attached to the surface of a coverslip, and a number of software are available to measure the chromatic shifts from such calibration samples. However, chromatic aberration can vary with depth, change with observation conditions and be induced by the biological sample itself, thus hindering determination of the true amount of chromatic shift in the sample of interest and across the volume. Correcting chromatic shifts at higher accuracy is particularly relevant for super-resolution microscopy where only slight chromatic shifts may affect quantitative analyses and alter the interpretation of multicolor images. We have developed an open-source software Chromagnon and accompanying methods to measure and correct 3D chromatic shifts in biological samples. Here we provide a detailed application protocol that includes special requirements for sample preparation, data acquisition, and software processing to measure chromatic shifts in biological samples of interest.

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.

Methods Article

超分辨率成像细菌科机械