Name: single plane illumination module and micro-capillary approach for a wide-field microscope
Uploaded: 2014-08-15T19:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

这个项目是由土地巴登 - 符腾堡州，以及欧洲联盟，的Europäischer全宗资助献给死去大区ENTWICKLUNG。作者感谢赖维蒂希（ILM乌尔姆）提供的U251-MG-L106-Grx1-roGFP2细胞系和克劳迪娅·欣策的熟练的技术援助。

1，细胞球体生长，育成<ol><li>实验1:细胞球体培养与化疗药物<ol><li>通过添加0.45克琼脂糖至30ml培养基中（足够用于6孔板）制备琼脂糖1.5％的培养基。 </li><li>加热该混合物从步骤1.1.1至至少80℃，同时从时间搅拌时间。 </li><li>补加入50μl从步骤1.1.2加热混合物到96孔板的每个孔中，并让在1-2小时之固化。储存关闭和覆盖在冰箱中于5℃下直到使用的板。 </li><li>种子约150 MCF-7乳腺癌细胞中所制备的96孔板，在Dulbecco改良的Eagle培养基（DMEM）和火腿的F-12培养基中补充有10％胎牛血清（FCS）和1％青霉素/链霉素的每个孔。 </li><li>在5％CO 2和37在培养箱中生长球状5-7天达到直径约为200-300微米76℃。 </li><li>孵育的细胞球粒用蒽环类抗生素多柔比星盐酸盐为6小时，其浓度在5％CO 2和37℃下2微米和8微米（在培养基中）在培养箱之间的范围内。 </li><li>洗细胞球状体与培养基或Earle氏平衡盐溶液（EBSS）于显微镜之前。 </li></ol></li><li>实验2:在球体表达氧化还原传感器的氧化过程<ol><li>种子约96孔板中在DMEM 400 U251-MG-L106成胶质细胞瘤细胞中永久地转染了谷胱甘肽敏感的绿色荧光的氧化还原传感器Grx1-roGFP2在琼脂糖凝胶包被的孔（在步骤中描述的步骤1.1.1-1.1.3）培养基补充有10％FCS，1％青霉素/链霉素和潮霉素B（150微克/毫升）。 </li><li>在5％CO 2和37℃下生长的球体5-7天达到直径约为200-300微米的培养箱。 </li><li>添加10＃181;将在12小时用升的过氧化氢溶液（purum PA，≥30％），以990微升双蒸水来创建100mM的储备液。 </li><li>在实验前才从步骤稀释原液1.2.3到50μm的EBSS。 </li><li>测量与过氧化氢50μm的EBSS为氧化还原传感器的氧化过程中孵育通过微流体系统的细胞球状体。 </li></ol></li><li>实验3:启动和球体内部细胞坏死的标签<ol><li> 96孔板的DMEM培养基中补充有种子约400个细胞（ 如 U251-MG-L106-Grx1-roGFP2胶质母细胞瘤细 ​​胞），在琼脂糖凝胶包被的孔（在步骤中描述的步骤1.1.1-1.1.3） 10％FCS，1％青霉素/链霉素和潮霉素B（150毫克/毫升）。 </li><li>在5％CO 2和37℃下生长的球体5-7天达到直径约为200-300微米的培养箱。 </li><li>孵育细胞球体与在1μM的在培养基中的浓度的线粒体电子传递抑制剂鱼藤酮3小时以诱导细胞坏死。另外，以1微升的1毫升培养基中的浓度共同孵育带有绿色的细胞毒性染料3小时以标记坏死细胞。 </li><li>用培养基或EBSS之前镜检洗细胞球体。 </li></ol></li></ol> 2，光表调整注意:为了达到最好的结果是，必须确保该光板的光束腰是在物镜的聚焦平面上。束腰的位置可以仅通过观察光片在垂直位置相对于毛细管对齐（参见步骤2.5 - 2.8）。 <ol><li>用倒置显微镜和安装SPIM激励模块，在参考文献中描述。 16，在定位表的底板（ 见图1A）。 </li><li>装备显微镜用10X / 0.3或20X / 0.5的显微镜物镜和一个适当的长通滤波器（ 例如，λ≥515纳米），在显微镜的检测路径。 </li><li>安装积分照相机的显微镜的检测端口。 </li><li>使用激光或激光二极管的并联的准直光束用的优选470nm处的激发波长和其应用到SPIM模块。 </li><li>通过90度时，其中光片传送到用于光片的光束腰的轴向调整垂直位置旋转的圆柱形透镜。 </li><li>将包含在样品架荧光染料液体的毛细管。 </li><li>附加的样本保持器与毛细管到显微镜的定位表和对准毛细管，使得它的中心和光板的光腰是在焦点的位置。 </li><li>通过柱面透镜本身的轴向位置的变化调整束腰。回头的广告后，镜头校正配料。 </li></ol> 3，细胞球体的应用和显微镜饲料同步<ol><li>单独或在长方形的硼硅玻璃毛细管16内组将细胞球粒用的600微米×600微米的内横截面和120微米的壁厚。两种技术的细胞球状体的应用已经被证明是成功的。 <ol><li>以空毛细管直立用拇指和中指，然后封好上口与你的食指。使下部开口附近的细胞球状体在其周围的液体（EBSS或培养基）。从上部开口松开食指。液体，在它的细胞球体将被毛细力立刻浸泡。调整填充的毛细管内的旋转椭圆体通过重力在毛细管的各直立位置的位置。 </li><li>备选地，通过移液应用细胞球体到毛细管。小心塔吨移液器的尖端的开口，一方面，对于球体的尺寸足够大，并且在另一方面，小于或等于在尺寸上与毛细管的内径。 </li></ol></li><li>将毛细管与球体它在显微镜特殊的样品架。 </li><li>附加的样本保持器与毛细管到显微镜的定位表和对齐细胞球状体，使得它的中心和聚焦的位置。 </li><li>集光片和显微镜物镜，在检测路径中，通过调整反射镜的位置和柱面透镜的聚焦平面之间的匹配（参见图1B中的调整螺钉）。 </li><li>调节测微螺钉（参见图1B）根据折射指数和物镜的数值孔径，以补偿鱼缸效果。 注:样品和周围吨媒体不同的折射指数他物镜引到物镜的移位和焦平面的移动之间的差异。由于光片被物镜转台移动时，光片（对应于所述物镜的位移）和焦平面的换档的换档之间的不匹配是通过一杆臂补偿（参见图1B）。测微螺钉用来调整上部和下部销之间的距离来调整光片的根据物镜的数值孔径和折射率的转变。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig1highres.jpg" width="500" /> 单平面照明的图1（A）的照片的单一平面照明模块安装于倒置显微镜的定位表的底板，（B）的设置模块和显微镜进料同步补偿焦平面和照明平面之间的升降失配（鱼缸效应）。 ΔZ○表示物镜和Δ狕的变速F表示焦平面的偏移和光片。嵌体显示毛细血管含有球状体的横截面。左:矩形毛细管600微米（墙120微米）的内径。右:用于旋转设置。的内圆毛细管（内径为400μm，外径为550微米）的外侧的矩形毛细管内转动。两个毛细管之间的空间填充有浸没流体。 4，测量的动态液体环境注:先前的协议之前，测量其中的细胞球状体已预先温育，然后保持在适当位置中的毛细管通过简单的重力用于静态孵育。没有必要进行进一步fixati上。然而，对于在流动介质，其中的动态孵化，因此，在动态环境时，优选测量吸收动力学的测量，就可以做到这一点的子协议。步骤4.5-4.9是关键避免气泡到达样品。 <ol><li>填充FCS的毛细管30分钟，以支​​持细胞球状体的存货细胞粘附到内玻璃表面上。 </li><li>让剩余的FCS涂层在贫毛细管干燥至少12小时。 </li><li>按照步骤3.2中所述引入细胞球体的FCS涂层毛细管。 </li><li>留在培养箱中的毛细管，在5％CO 2和37℃下进行另外的2-4小时，以使球体的细胞粘连。 </li><li>设置了微流体系统的汇流部（参见图2）。使用蠕动泵以填充管的汇流（内径:0.89毫米），含有荧光染料，药物或试剂的培养基中。 </li><li>连接到打印机克拉微流体系统来的气泡捕捉器的汇流部（参见图2）。 </li><li>第一夹紧毛细管无气泡的汇流导管，然后将毛细管的另一端夹在排液管。 </li><li>调的水浴中以所希望的值（ 例如，37℃）的液体的温度。 </li><li>调节蠕动泵以期望的泵速度（ 例如，流速:9微升/分钟;泵速度在毛细管:25毫米/分钟）。 </li><li>收集泵送液体无论是在收件人（开环设置），反馈给源（闭环设置），或者直接将其送到蠕动泵（紧闭环设置）的管道。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig2highres.jpg" width="500" /> 图2微流控设置（开环和紧闭环）;镶嵌:照明用的SPH使用连接到一个倒置显微镜光片以荧光显微镜微毛细管内eroid。 5，数据采集与分析<ol><li>设置激光功率和积分时间的图像采集。 </li><li>照顾，在步骤5.1中定义的参数不超过光的剂量值约为50-100焦耳/ cm 2的天然细胞或10-20焦耳/ cm 2的细胞培养用荧光染料或转染的荧光蛋白编码质粒来避免光毒性（详见参考文献12）。 </li><li>设置增量为Z堆叠到Δ狕= 5-10微米是优选的3维数据分析作为光片厚度为约10微米。 </li><li>由细胞球体内变化的焦平面的执行单个图像或Z-堆栈的测量。 </li></ol>

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本手稿描述光片或单一平面照明显微镜（SPIM）装置，其3维电池系统进行了优化， 例如，多细胞肿瘤球体（MCTS）。三个示例性的应用包括:（1）吸收细胞生长抑制药物和其部分转化为降解产物（其对化疗疗效贡献仍有待评估）中，氧化还原状态的（2）通过采用遗传编码谷胱甘肽传感器在测量另外一种氧化剂，和（3）启动和细胞坏死时抑制线粒体呼吸链的标记。 本SPIM?...

除了 ​​公认的方法（共焦或多光子激光扫描显微术1-4，结构照明显微镜5,6）光片或单个平面照明显微镜（SPIM）已被证明是三维成像7,8的一种有价值的方法， 9。特别令人感兴趣的是其应用到三维细胞培养物， 例如，多细胞肿瘤球体（MCTS），它们越来越多地用于药物发现研究10,11。再者，SPIM是当即使在长期暴露或重复测量低光剂量必须保持样品的可行性，因为用于样品仅此平面被曝光的每个平面的测定的优先方法。这是相对于其他显微技术，其中对于检测各焦平面的整个样品被照射，使记录的许多平面时的光量总和起来，并可能损坏样品12。 </P&gt; 光片显微镜或SPIM是根据与观察路径的样品在垂直方向上或者通过使用圆柱形透镜的或者通过扫描激动人心的激光束（用于见参考文献8评论）的照明。这通常需要特殊的样品室13,14或矩阵， 例如，琼脂糖7,15，在特殊的高性价比的显微镜来实现。作为替代那些系统中可比简单的照明装置，SPIM已经开发并适于常规的倒置显微镜16（参见图1）。它由激光束扩大到直径为8毫米，集中由一个圆柱形镜头（焦距:50毫米，数值孔径:0.08），以6-10微米厚的片状光通过景深约100微米。样品位于600至900微米内径的矩形毛细管放置在显微镜物镜的len的前S代表荧光检测。这些主要功能是目前完成，通过使用先进的微毛细管的方法，用于保持和转动用于荧光检测（相同光路中的样本，照射光片的同步调整（在轴向）和物镜的优化位移的长度，所需要的机械进给的校正），和一微流体系统的适配为应用荧光染料，药物制剂或药物，从而减少所需的数量和费用。

<table><tbody><tr><td>Microtiter plate</td><td>Orange Scientific</td><td>4430100</td><td>For cell spheroid growing</td></tr><tr><td>Agarose&amp;nbsp;</td><td>Carl Roth GmbH</td><td>3810.1</td><td>For cell spheroid growing</td></tr><tr><td>MCF-7 cell line</td><td>CLS Cell Lines Service GmbH</td><td>300273</td><td>Cell line</td></tr><tr><td>U251-MG-L106 cell line</td><td></td><td></td><td>Cell line</td></tr><tr><td>DMEM</td><td>Biochrom AG (Merck Millipore)</td><td>FG0435</td><td>Culture medium</td></tr><tr><td>DMEM/Ham&#039;s F-12</td><td>Biochrom AG (Merck Millipore)</td><td>FG4815</td><td>Culture medium</td></tr><tr><td>FCS</td><td>Biochrom AG (Merck Millipore)</td><td>S0615</td><td>Cell culture supplement</td></tr><tr><td>Penicillin/streptomycin</td><td>Biochrom AG (Merck Millipore)</td><td>A 2213</td><td>Antibiotics</td></tr><tr><td>Hygromycin B</td><td>PAA Laboratories&amp;nbsp;&amp;nbsp;</td><td>P02-015</td><td>Antibiotic</td></tr><tr><td>EBSS</td><td>Sigma-Aldrich Inc.</td><td>E3024</td><td>Cell culture supplement</td></tr><tr><td>Doxorubicin</td><td>Sigma-Aldrich Inc.</td><td>D1515</td><td>Fluorescent dye (CAUTION: acute toxicity)</td></tr><tr><td>Green cytotoxicity dye</td><td>Promega GmbH</td><td>G8742</td><td>CellTox - fluorescent cytotoxicity dye</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich Inc.</td><td>R8875</td><td>Cellular inhibitor (CAUTION: acute toxicity)</td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich Inc.</td><td>95302</td><td>Reagent for oxidation (CAUTION: acute toxicity)</td></tr><tr><td>Capillary</td><td>VitroCom&amp;nbsp;</td><td>8260-050</td><td>Sample preparation</td></tr><tr><td>Microscope</td><td>Carl Zeiss Jena</td><td>Axiovert 200M</td><td></td></tr><tr><td>AxioCam MRc CCD-camera</td><td>Carl Zeiss MicroImaging GmbH</td><td>426508-9901-000</td><td>CCD-camera</td></tr><tr><td>AxioVision data aquisition software</td><td>Carl Zeiss MicroImaging GmbH</td><td>version 4.8.2.</td><td></td></tr><tr><td>Laser diode</td><td>Pico Quant GmbH</td><td>LDH-P-C-470</td><td>Used with driver PDL800-B</td></tr><tr><td>Peristaltic pump</td><td>Ismatec Labortechnik</td><td>MS-1 Reglo</td><td>Re-calibrated to reduce the minimum pump speed by 1/10&amp;nbsp;</td></tr><tr><td>Silicone tubes</td><td>IDEX Health &amp;amp; Science GmbH</td><td>TYGON R3607</td><td></td></tr></tbody></table>

single plane illumination module and micro-capillary approach for a wide-field microscope

这是很容易适应于一个倒置的宽视场显微镜和三维细胞培养物的优化，对于光片或单个平面照明显微镜（SPIM）中描述的模块， 例如，多细胞肿瘤球体（MCTS）。在SPIM激发模块的形状和偏转，使得样品被光片照射垂直于显微镜的检测路径的光。该系统的特点是利用一个矩形毛细管的用于保持（和在一个先进的版本也被用于旋转微毛细管的方法）的样品中，由照明光片的同步调整并用于荧光检测以及物镜通过微流体系统的应用的荧光染料，在少量的药物制剂或药物的适应性。给出的协议与该系统的工作，和一些技术细节的报告。代表性的结果包含的向上（1）的测量在添加氧化剂的需要抑制细胞生长的药物（阿霉素）和其部分转化为降解产物，（2）氧化还原测量通过使用遗传编码谷胱甘肽传感器，以及（3）启动和细胞坏死的经抑制的标签线粒体呼吸链。本SPIM模块与现有系统相比，差别和优点进行了讨论。

实验1:细胞球体培养与化疗药物 预先培养的MCF-7细胞球体（8μM多柔比星，6小时）的Z堆叠的扫描示于图3，它给出了关于细胞摄取和多柔比星17的分布和其降解产物18,19的详细信息。内的旋转椭圆体的红色荧光阿霉素的外细胞层主要定位于细胞核，而在内侧旋转椭球体的区域由降解产物发射绿色荧光变为主导的细胞膜19。 <p cl...

Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

为单一平面照明显微镜的模块（SPIM）中描述了很容易适应的倒宽视场显微镜和3维细胞培养物进行了优化。样品位于一个矩形的毛细管，并通过微流体系统的荧光染料，药物制剂或药物可以以小批量应用。

单一平面照明模块和一个宽场显微镜微毛细管法

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

single-plane-illumination-module-micro-capillary-approach-for-wide

Research

JoVE Journal

Engineering

9.7K 次观看.  Aalen University. 为单一平面照明显微镜的模块（SPIM）中描述了很容易适应的倒宽视场显微镜和3维细胞培养物进行了优化。样品位于一个矩形的毛细管，并通过微流体系统的荧光染料，药物制剂或药物可以以小批量应用。 

视频: Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

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

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

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

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

高分辨率光纤内窥镜为原位细胞成像

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

科研

生物工程

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 ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

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 ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

生物化学

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

免疫与感染

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

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 ...