这项工作是由年轻的研究员项目 (NRF-2015R1C1A1A02036464), 工程研究中心计划 (NRF-2015R1A5A1037668) 和全球前沿计划 (CAMM-2014M3A6B3063708), m.k, 轮, I.K. 承认全球博士学位资助。奖学金 (NRF-2017H1A2A1043204、NRF-2017H1A2A1043322、NRF-2016H1A2A1906519) 通过韩国国家研究基金会 (NRF) 资助, 由韩国政府科学、信息和通信技术和未来规划部 (MSIP) 提供。

1. 基板制备 <ol> <li> 获得高度精炼的石英晶片。对于在这里报告的制造, 使用500和 #181; m 厚度的硅片. </li>
	<li> 自旋涂层石英晶圆片与正光刻胶在 2000 rpm 和烘烤六十年代在90和 #176; C. 
	注: 正光刻胶层涂层, 以防止在随后的切割步骤损害. </li>
	<li> 使用划片机将光刻胶切成小块 20 x 20 mm 2 大小. </li>
	<li> 用压缩的氮气枪吹掉切割步骤产生的微粒. </li>
	<li> 在去 (DI) 水中的超声波浴中放置5分钟, 在45和 #176; c. 用丙酮超声波浴去除光刻胶层5分钟, 在45和 #176; c. 用两个超声波浴, 丙酮和异丙醇清洁基板, 每在5分钟45和 #176; C. </li>
	<li> 用压缩的氮气枪将基体干燥. </li>
</ol> 2。蚀刻掩码图案 <ol> <li> 将清洁的石英衬底装入真空电子束蒸发系统。确保基板旋转已启用. </li>
	<li> 沉积率为2和 #197 的铬层;/s. 
	注: 在蚀刻掩模上应放置至少100纳米厚度的层, 以防止由沉积物制成的针孔. </li>
	<li> 按下排气口按钮以释放腔室, 并使用导电铜带将样品安装在聚焦离子束 (纤维蛋白原) 支架上. </li>
	<li> 将心室支架装入心室室. </li>
	<li> 关闭会议厅门并按下泵按钮以疏散会议厅. </li>
	<li> 选择和 #34; 横梁和 #34; 在光束控制卡舌下, 将离子束电流 (7.7 pA) 和加速度电压 (30 伏) 设置为心室模式. </li>
	<li> 打开离子束系统. </li>
	<li> 选择和 #34; 横梁和 #34; 在 "光束控制" 选项卡下打开电子束, 并使用 "低放大率" 软件聚焦图像. </li>
	<li> 在扫描电子显微镜 (SEM) 模式下的导航标签下设置工作距离 (WD) 4 毫米. </li>
	<li> 将支架的倾斜角度设置为52和 #176; 在孔阵列掩模制作前, 以不同放大的扫描电镜图像. </li>
	<li> 在 "图案" 选项卡下, 选择图案区域并在铬层上制作一个 50 nm 孔阵列. 
	注意: 在 "模式" 选项卡下可以访问简单的模式工具。通过导入位图或生成脚本, 可以实现更复杂的几何和曝光控制. </li>
	<li> 完成后, 关闭电子束和离子束系统, 冷却系统. </li>
	<li> 按下排气口按钮, 用氮气将燃烧室排出。把持有者带出会议厅. </li>
	<li> 关闭会议厅门并按下泵按钮以疏散会议厅. </li>
</ol> 3。湿法蚀刻工艺和去除掩膜层 <ol> <li> 将图案基板放入1:10 缓冲氧化物蚀刻中 5 min. 
	注: 石英是有选择和各向同性湿蚀刻的蚀刻和形成一个球形的形状。所用蚀刻掩模可获得透镜的形状, 其直径由蚀刻时间精确控制。一个更好的球形形状可以形成一个较小的图案直径。1.5 和 #181; m 直径半球可在5分钟内获得. </li>
	<li> 将图案衬底放入 DI 水中以清洗缓冲氧化物蚀刻 (5 分钟, 2 次). 
	注: 缓冲氧化物蚀刻可能是危险的, 所以使用此蚀刻时要小心. </li>
	<li> 用压缩氮气干燥样品. </li>
	<li> 将图案衬底放入 CR-7 铬蚀刻中以除去铬面膜层. 
	注: 除去铬层后, 可获得直径为1.5 和 #181 的球形衬底. </li>
	<li> 将图案基片放入 DI 水中以进行清洁 (5 分钟). </li>
</ol> 4。多层沉积和纳米物体的题字 注意: 在球面石英衬底上放置一对层。在这里, Ag 和 2 被用作沉积材料。Ag 和 2 在厚度为 15 nm 的情况下交替沉积. 
<ol> <li> 按下电子束蒸发系统的排气按钮, 然后等待通风口结束. </li>
	<li> 将图案的基板装入真空电子束蒸发系统后的排气口. </li>
	<li> 关闭会议厅门并将真空度疏散到 10 -7 乇通过按下泵按钮. 
	注: 真空状态应保持在 10 -7 乇, 以减少表面粗糙度的散射. </li>
	<li> 将1和 #197 的增长率存入银层, 并沉积15纳米厚的银层. </li>
	<li> 在 Ag 层的沉积之后, 冷却基体为 5 min. </li>
	<li> 改变电子束蒸发系统的口袋, 选择另一个坩埚, 并存放在 2 层的增长率为1和 #197;/s. 存入 15 nm 厚的包 2 层. 
	注: 在沉积过程中, 薄膜的生长速率保持在较低的水平, 以维持表面粗糙度的均匀性. </li>
	<li> 在沉积后的 "2" 层, 冷却基板为5分钟. </li>
	<li> 重复步骤 4.4-4.7, 为数十个周期存入一个多层 Ag 和. 2 . 
	注意: 在这一点上, hyperlens 的制作已经结束。下一步是为测试 hyperlens 成像能力而制作任意 sub-diffraction 限制的功能。纳米孔和狭缝是由纤维蛋白原铣削. </li>
	<li> 改变电子束蒸发系统的口袋, 并将铬层沉积在50纳米的厚度上. </li>
	<li> 铬层沉积后, 关闭电子束蒸发系统。按下排气按钮, 通过引入氮气来排出腔室. </li>
	<li> 在通风口后, 打开舱门, 将支架带出会议厅。脱下制作的 hyperlens 装置. </li>
	<li> 关闭会议厅门并按下泵按钮以疏散会议厅. </li>
	<li> 根据制造商和 #39 的说明, 将含铬的 hyperlens 装入到 "纤维蛋白原" 铣削系统和图案的纳米结构中. </li>
</ol> 5。设置成像系统和成像过程 <ol> <li> 将常规传输型光学显微镜放在光学表上. 
	注: 在这里, 用倒置光学显微镜作为主体. </li>
	<li> 使用适配器将白光光源连接到显微镜照明路径. </li>
	<li> 放置一个以 410 nm 为中心的光学带通滤波器. 
	注: 带通滤波器有选择地穿透特定波长的光;在这里, 410 nm 光照亮了样品。由 Ag 和 2 组成的 hyperlens 具有 410 nm 波长的高性能。仿真结果 ( 图 2c ) 显示了 hyperlens 的性能, 它满足了 410 nm 光下的双曲色散关系. </li>
	<li> 选择一个高倍油浸物镜。使用高质量的 CCD 摄像机获取图像. 
	注意: 这个光学设置只是把禁令dpass 过滤到光照明路径, 以排序出 410 nm 波长光。在不使用白光的情况下, 可以在样品上照射特定波长的光, 但在普通的实验室中, 光学显微镜可以通过亮场或荧光成像来观察样品的白光源. </li>
	<li> 在物镜上放置一滴浸入式油。在采样阶段放置一个 hyperlens 并捕获图像. 
	注: 在 hyperlens 的内表面上刻有纳米级的物体可以用410纳米光照亮。随着 hyperlens, 纳米级的物体将被放大并被物镜捕捉, 并被 CCD 摄像机所成像. </li>
</ol>

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作者声明他们没有竞争的金融利益。

hyperlens 的制作包括三主要步骤: 通过湿蚀刻过程将半球形几何定义为石英基底, 用电子束蒸发系统堆叠金属和介电多层, 并刻在 Cr 层上的对象。最重要的步骤是第二, 因为它可以显著地影响质量的 hyperlens。在薄膜沉积过程中, 有两个条件需要特别注意清晰的构图像。堆叠多层形是其中的一个关键问题, 由于 non-conformal 沉积的多层导致一个偏离完美的球形形状。如果薄膜沉积速度不够慢, 在中心和半?...

在活细胞中观察生物分子的愿望导致了显微镜的发明, 显微镜的问世在过去的几个世纪里传播了生物学、病理学和材料科学等各个领域的革命。然而, 进一步的研究进展受到了衍射的限制, 这限制了常规显微镜的分辨率约为波长的一半1。因此, 克服衍射极限的超分辨率成像是近几十年来的一个有趣的研究领域。
由于衍射极限是由于倏逝波的损失, 其中包含波长信息的对象, 早期研究已经进行, 以保持倏逝波消失或恢复他们2,3。为了克服衍射极限的努力, 首次报道了近场扫描光学显微镜, 它收集的倏逝场在接近对象之前, 它被驱散2。然而, 由于扫描整个图像区域和重建需要很长的时间, 它不能用于 real-time 成像。虽然另一种基于 "透镜" 的方法, 它放大了倏逝波, 提供了 real-time 成像的可能性, 但波长成像只能在近场区域中进行, 无法远远超过对象4,5,6,7。
最近, hyperlens 已成为一种新的方法来 real-time 远场光学成像8,9,10,11,12。hyperlens 是由高各向异性双曲型材料13组成的, 它具有平坦的双曲色散, 因此它支持具有相同相速度的高空间信息。此外, 由于动量守恒定律, 高横向波随着波通过圆柱形几何而逐渐压缩。这种放大的信息可以通过远场区域的传统显微镜来检测。这对于 real-time 远场成像是特别重要的, 因为它不需要任何定点扫描或图像重建。此外, hyperlens 可以用于除成像以外的其他应用, 包括纳米。由于反对称14、15、16, 通过反向方向的 hyperlens 的光线将聚焦到 sub-diffraction 区域。
在这里, 我们报告的球状 hyperlens, 放大 two-dimensional 信息在可见光的频率。与传统的圆柱形几何不同, 球形 hyperlens 在两个侧面尺寸中放大物体, 从而促进实际的成像应用。详细介绍了 hyperlens 的制作方法和成像装置, 并对高质量的 hyperlens 进行了详细的再现。为了证明它的 super-resolving 力, 波长的物体被刻在 hyperlens 上。证实了被 hyperlens 的小特征被放大。因此, 在远场区域中实时地得到了清晰解析的图像。这种新型的球形 hyperlens, 其易于与传统的显微镜结合, 提供了实际的成像应用的可能性, 导致了一个新的时代, 生物学, 病理学和一般纳米。

<table><tbody><tr><td>Focused Ion Beam milling machine</td><td>FEI</td><td>Helios Nanolab G3 CX</td><td></td></tr><tr><td>E-beam evaporation system</td><td>Korea Vacuum Tech</td><td>KVE-E4000</td><td></td></tr><tr><td>Scanning electron microscopy</td><td>Hitachi</td><td>SU6600</td><td></td></tr><tr><td>Inverted microscopy</td><td>Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Light source</td><td>EXCELITAS Technologies</td><td>X-Cite 110 LED</td><td></td></tr><tr><td>Band pass filter</td><td>Chroma</td><td>ET405/30M</td><td></td></tr><tr><td>Objective lens</td><td>Zeiss</td><td>Plan-Apochromat</td><td>NA=1.3, 100X</td></tr><tr><td>CCD camera</td><td>Andor</td><td>Zyla 4.2</td><td></td></tr><tr><td>Quartz wafer</td><td>CORNING</td><td>Fused Silica Corning 7980</td><td></td></tr><tr><td>Buffered oxide etchant</td><td>J.T Baker TM</td><td>J.T.Baker 5175</td><td></td></tr><tr><td>Photoresist</td><td>AZ electronic materials</td><td>GXR-601 PR</td><td></td></tr><tr><td>Chromium etchant</td><td>SIGMA-ALDRICH</td><td>651826</td><td></td></tr><tr><td>Aceton</td><td>J.T Baker TM</td><td>UN1090</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>J.T Baker TM</td><td>UN1219</td><td></td></tr><tr><td>FEM simulation tool</td><td>COMSOL 5.1 Multiphysics</td><td></td><td></td></tr></tbody></table>

demonstration of a hyperlens-integrated microscope and super-resolution imaging

利用超分辨率成像克服传统显微镜的衍射极限, 引起了生物学和纳米技术研究人员的兴趣。虽然近场扫描显微镜和 superlenses 改善了近场区域的分辨率, 但远场成像在 real-time 仍然是一个重大挑战。最近, 将倏逝波放大并转化为传播波的 hyperlens, 已经成为远场成像的一种新方法。在这里, 我们报告的制造一个球形 hyperlens 由交替银 (银) 和氧化钛 (2) 薄层组成。与传统的圆柱形 hyperlens 不同, 球面 hyperlens 允许 two-dimensional 放大。因此, 纳入常规显微镜是直接的。提出了一种新的与 hyperlens 集成的光学系统, 使得在远场区域能实时获得波长图像。在本研究中, 详细说明了制作和成像的设置方法。这项工作还描述了 hyperlens 的可及性, 以及 real-time 成像在活细胞中的实际应用, 这可能导致生物学和纳米技术的革命。

hyperlens 装置解决 sub-diffraction 特性的能力依赖于其均匀性和高质量的制造。在这里, 一个 hyperlens 是由一个多层的 Ag 和总2交替存入。图 2a显示了制作精良的 hyperlens17的 SEM 图像。横断面图像显示, 在半球石英衬底上沉积有均匀厚度的 Ag 和 Ti3O5薄膜的多层膜。最终 hyperlens 结构的表面粗糙度小于 1.5 nm 的根均方?...

Watch this Scientific Journal Video about Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging at JoVE.com

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

hyperlens 的应用是一种新型的超分辨率成像技术, 由于其在 real-time 成像中的优越性和常规光学的简单实现。在这里, 我们提出了一个描述球面 hyperlens 的制作和成像应用的协议。

Hyperlens 综合显微镜和超分辨率成像的演示

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

demonstration-hyperlens-integrated-microscope-super-resolution

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

7.7K 次观看.  Pohang University of Science and Technology (POSTECH). hyperlens 的应用是一种新型的超分辨率成像技术, 由于其在 real-time 成像中的优越性和常规光学的简单实现。在这里, 我们提出了一个描述球面 hyperlens 的制作和成像应用的协议。

视频: Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

片上的无透镜荧光显微镜

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

科研

生物工程

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

免疫与感染

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

发育生物学

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Here we describe a photobleaching method to reduce the autofluorescence of cyanobacteria. After photobleaching, stochastic optical reconstruction microscopy is used to obtain three-dimensional super-resolution images of the cyanobacterial FtsZ ring.

光漂白可实现前列腺素球菌中 ftsz 环的超分辨率成像

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

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 ...

生物学

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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 ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.