Name: preparing adherent cells for x-ray fluorescence imaging by chemical fixation
Uploaded: 2015-03-12T13:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation at JoVE.com

The authors acknowledge Stefan Vogt for his assistance in the fitting of the representative data shown in this paper, and helpful discussions. The authors also acknowledge Chris Jacobsen for his support to Q. J. 
Use of the Advanced Photon Source, beamlines 2-ID-E and 8-BM-B, at Argonne National Laboratory was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

1.制备仪器，基板，文化传媒和菜<ol><li>处理氮化硅（Si 3 N 4）的窗口。 <ol><li>略通过挤压一端包含窗口以这样的方式，以不挤窗口本身打开胶囊，一边旋转用另一只手的胶囊的另一端。 </li><li>检查一对反向的，体视显微镜下的细尖的镊子，以确保有在尖端无粘性，缺口或弯曲。否则，该窗口可以由镊子破裂或断开他们的飞在随后的步骤。 </li><li>通过仔细地抓用镊子窗口的帧删除从胶囊的窗口中，同时轻轻挤压胶囊靠近开口端施加压力的方向，以使上述胶囊更宽其中窗口的边缘需要出来。 </li></ol></li><li>窗口预洗（可选）。 <ol><li>如果需要，预洗窗轻轻蘸他们入蒸馏水，持续5秒，然后2分钟洗涤在70％乙醇和2分钟的洗涤在100％乙醇。 </li></ol></li><li>粘贴并在培养皿灭菌窗户。 <ol><li>贴上窗，设置平面朝上在培养皿底部的窗口中，将一块胶布上干净的载玻片上。削减约四分之一英寸的带断下带的长边。取的胶带片断的短边（在⅛"由¾"）配有一个干净的刀片。 </li><li>使用镊子的胶带片应用到电网或窗口的边缘。使用足够安全地坚持它不涉及窗口开放本身。操纵粘附胶带用镊子，向下设置窗口到培养皿的底部。使用镊子的顶端附着胶带到培养皿牢固的底部。 </li><li>适用的另一种带条的另一边缘和粘附这对底牢固地用镊子的尖端。 </li><li>一旦所有的窗户都录音，消毒在培养皿窗用紫外线（UV）辐射。通过将盘在紫外灯下在层流罩约1小时完成此。 </li><li> （可选）如果窗口是被预先涂有聚赖氨酸，这样做如下窗口灭菌。与培养基外套与10微升的无菌0.01％聚-L-赖氨酸1小时在37℃培养箱溶液，然后在窗口冲洗剩余溶液关闭。 </li></ol></li><li>准备缓冲区XFM前的最后洗净。 <ol><li>制备任一个的Tris-葡萄糖或哌嗪-N，N&#39;-双（乙磺酸）（PIPES）缓冲液-sucrose不含任何痕量金属与渗透压和pH相当于Dulbecco磷酸盐缓冲盐水（D-PBS），无钙和镁。使用这些缓冲区之一冲洗细胞细胞化学固定后。 <ol><li>使的Tris-葡萄糖溶液（10mM的Tris，260 mM的葡萄糖，9毫乙酸）添加1.4克葡萄糖，36毫克的Tris碱和16微升乙酸到20ml的超纯水。然后，调节最终体积至30ml，用超纯水。不需要进行pH调节。存储在室温至一个星期。 </li><li>为了使PIPES-蔗糖（20毫摩尔PIPES，200mM的蔗糖）添加0.18克PIPES和2.0g蔗糖到20ml的超纯水和将pH调节至7.4与少量浓乙酸。然后，调节最终体积至30ml，用超纯水。存储在室温至一个星期。 注:原因，以避免在最后的漂洗用PBS是磷酸盐，氯离子和钾在PBS中的浓度是如此之高，它可以与XFM干扰，如果它没有完全空气干燥前除去。 </li></ol></li></ol></li><li>准备缓冲区细胞接种。 <ol><li>制备或采取了所有媒体，如RPMI培养基1640，1×胰蛋白酶-EDTA溶液，D-PBS，并根据需要对特定粘附细胞系作为通常用于传代的粘附细胞ø作出的任何其他试剂˚F兴趣，温暖他们到37℃。 </li></ol></li></ol> 2.电镀细胞<ol><li>含有该窗口，在层流罩中无菌培养皿中，轻轻地添加介质（10毫升100毫米培养皿），沿着所述培养皿用它倾斜成一角度的侧面。避免产生不必要的表面张力，或直接拖放到媒体的氮化硅窗户表面。媒体被添加，​​慢慢解除的倾斜角以涂覆网格和窗口介质。 </li><li>准备感兴趣的贴壁细胞系适当，胰蛋白酶和刮细胞脱落板像往常一样的传代细胞系。应用细胞的新鲜板之前执行的任何细胞计数，或其他必要的准备。 </li><li>加入细胞培养皿与窗户是需要的通常在24-72小时达到50％-70％汇合的细胞密度，并在湿润的培养箱中5％二氧化碳孵育在37℃。 </LI&gt; <li>观察细胞的生长偶尔，使用倒置显微镜，直至所需的细胞密度（通常为50％-70％汇合）为止。 </li></ol> 3.固定细胞<ol><li>通过稀释所购买的股票（如25％多聚甲醛）并调节pH至7.4，用乙酸（以避免加入过量的钠的样品）制备在D-PBS新鲜的4％多聚甲醛。 </li><li>当细胞已生长如需要的话，和任何重要污渍（如赫司特）已经被施加并成像，通过温和抽吸取出介质，倾斜盘以45°的角度，用一只手，并用手移液管与其它吸。 注意:不要用移液介质的，另一种是选择窗口起来，蘸窗口，进入离心管与D-PBS两次，然后将其放置到一个新的培养容器与固定液解决方案，20分钟后洗净PBS的2倍以去除残留固定剂。如果选择了此项，去DIRectly步骤3.5。 </li><li>轻轻冲洗菜D-PBS，并轻轻加入新鲜的4％PFA / PBS同时举行的菜在45°角，移液对菜的一侧，然后缓慢放松的倾斜角度，使立即更换去除液体固定剂溶液以覆盖窗口。保持覆盖有固定剂为20分钟，在室温将细胞。 </li><li>轻轻吸去除液体，再上面，倾斜的菜稍微用手吸。 </li><li>轻轻加入一些管道/蔗糖溶液中，使用步骤3.3中所述的倾斜和移液的方法重新装上卸下液体。 </li><li>重复步骤3.4和3.5。 </li><li>准备材料干燥。 <ol><li>收集无绒布毛巾等的Kimwipes和拉一开箱所以它是非常方便快捷和容易。 </li><li>准备一个干净，无级的地方设置窗口晾干。设置在电子显微镜中使用的那种，有脊等的橡胶垫格该窗口可以在一端支撑，而它干燥。这将保持窗口从卡住至干燥表面，因为它干燥时，和漏任何剩余缓冲器到样品的边缘。 </li></ol></li><li>仔细tweeze附连到窗口保持窗口向上出来的液体的胶带。检索窗户脱落带的通过施加少量PIPES /蔗糖，让他们浮起并然后拾取它们与反向镊子。 </li><li>小心（不接触窗口本身），并彻底地涂抹从用Kimwipe窗口的边缘的任何多余的PIPES /蔗糖。也涂抹用的Kimwipe的圆形折回来缩进区域。 注意:我们的目标是有盐或缓冲剂，作为小结晶窗口尽可能的表面上。 </li><li>把窗口拖到网格垫。确保该窗口未在网格垫平放，但撑起上的边缘（使用网格垫的脊）和触摸尽可能少的网格的垫越好。让窗口干燥。 </li></ol> 4.样品安装和成像<ol><li>一旦样品已干燥，验证细胞对使用光学显微镜的窗口的存在。执行此验证，准备样品运输到同步之前。 </li><li>打包窗口运输同步加速器光束线的托管实验。轻轻贴上窗户用胶带上双方向载玻片，然后贴上带有胶带的载玻片于塑料培养皿的底部。用胶带将培养皿关。 注:在该点之前，这些步骤可以以任何典型的实验室中进行。下面的步骤将最擅长的同步加速器光束线来完成。 </li><li>从轻轻用镊子窗口中删除的磁带。用指甲油，在沿窗口的边缘的薄均匀涂层，以固定的窗口由束线合作者在同步加速器提供的铝支架。 </li><li>将铝架成的运动装入，然后将其放入在X射线显微镜的位置。安装在其上的束线连接到机动阶段，样品腔室内部的支架上的样品。 </li><li>退出后，将样品透视镜仪器区（铅做的填充墙一厨），设置扫描。使用束线专用软件，选择的样品进行扫描的适当区域，同时用配有视频十字形和照相机观察在样品上的X射线束的位置的预对准的下游闪烁照相机。 </li><li>选择适当的分辨率的样品的基础上，所关心的最小特征的尺寸的估计。对于单一的成像哺乳动物细胞（〜20-40微米大小），用0.25-0.5微米的分辨率。用于成像的组织的区域，且彼此可区别的细胞层，用2-20微米的分辨率。 </li><li>选择适当的停留时间为样品，基于所述满足量的估计值人的利益存在。对于一个快速浏览扫描，或者高量金属的存在，使用飞扫描与每像素30-300毫秒的停留时间。如果小料存在，或测量低相对丰度的金属，使用分步扫描与每像素1-2秒的停留时间。 </li><li>程序扫描成束线专用软件。获得光栅扫描图像。与光束线合作者一起工作，分析软件中的数据，将确定的特征能量峰的每一个元素。 </li></ol>

The authors have nothing to disclose.

在许多领域，包括地球科学，材料科学，化学生物学26-34 X射线荧光成像是非常有用的。进展同步辐射X射线，和它们的聚焦，产生了非常高的强度的光束。聚焦X射线束足以激发内源的金属中的细胞现在存在，产生可与目前可用的硅漂移检测器技术进行测量的信号。和学习金属的化学生物学中，细胞是在特定的一个应用程序，利用许多近期的发展在这方面的。 然而，使...

X射线荧光成像可以用于两者的身份和样品中存在的元素的量，以在空间上分辨。选择为大于电子结合感兴趣的重元素的能量的能量的入射X射线，克服内壳层电子的结合能的核1。这会在电子层一个"洞"。作为高能量电子落入到这些孔中，荧光X射线被发射的，其波长依赖于那些轨道的能量的分离。由于轨道的能量间距是一个给定的元件的特性，在X射线荧光发射也具有特征波长，取决于在元件上。正是这种发射在特征波长，允许存在的元素的识别。的荧光强度的校准允许存在的元素的定量分析。 X射线荧光microscopY（XFM）已经越来越使用，部分原因是非常辉煌的X射线同步辐射光源，例如那些在春天-8在日本，欧洲同步加速器辐射设备（ESRF）在法国，以及先进光子源（发展APS），在美国的2。这些源提供非常高强度X射线束。同时，改善在透视光学，如带片的技术，允许这些光束到亚微米的斑点的聚焦，虽然相当低效3。具有非常高强度的光束，即使是相对小的光量，可以集中足以激发内源的金属中的细胞，产生的信号，可以与目前可用的检测器技术来测量。因此，研究金属的化学生物学中，细胞是在特定的一个应用程序，利用许多的最近发展的在该技术4-10。 存在要考虑的，而应用程序的许多关键因素卧XFM调查培养的哺乳动物细胞或其它生物样品的元素分布和定量。首先，将样品需要保持完好，在结构上并且相对于它的元素组成中，为了进行测定才有意义。其次，样品也必须以某种方式保存，使得它是耐寒到能够通过聚焦X射线束而引起的辐射损伤。该样品可以一次同时满足这些标准的一种方法是将快速冷冻成玻璃状，无定形冰11,12。快速冷冻往往是通过诸如暴跌冻结或高压冻结13-16各种冷冻保存技术来实现的。人们普遍认为冻存保留整体蜂窝结构，化学成分的生物样品尽可能接近天然状态为可能的。化学固定，另一方面，由于缓慢和选择性渗透固定剂进入细胞和组织如瓦特ELL在膜渗透性作为以后的变化，可以允许各种细胞的离子特别是可扩散的离子如Cl，Ca和K至浸出，丢失或重定位，从而使这些元件不理想的17-19的调查。尽管低温定影超过化学固定在一般情况下，对于贴壁哺乳动物细胞，尤其是明显的优势，冷冻保存有各种限制20-23。其中最明显的是，并不是每一个研究实验室可方便前往冷冻保存仪器。大多数当前的高压冷冻甚至沉浸冷冻费用昂贵，拥有仅由冷冻设施的子集，其可以远离其中细胞温育。冷冻保存的好处可能被交易的出行压力置于细胞的缺点。因此，尽管冻存肯定是最严格的方式来保存样品X射线荧光分析，它肯定不是最方便的在各种情况下所有的研究人员;也不总是必要 - 如果感兴趣的金属是紧密地结合到可定影大分子，并且在该样品将被成像的分辨率大于在干燥过程中可能出现的超显微组织的损害。注意到的告诫24的，化学固定和干燥可以是合适的选择。 其他因素在成功的X射线荧光成像实验，包括适当的分析。 X射线荧光成像是从根本上的X射线荧光发射光谱结合光栅扫描，以提供空间分辨率。收集到的X射线荧光发射光谱重叠的包含发射峰，背景和入射光束的弹性和非弹性散射峰的组合。软件，使反卷积的这些贡献和排放峰值的拟合，得到了关键的发展，这一领域的25。此外，开发和商业DIST已知成分，用于校准相对于物质量荧光强度薄膜标准ribution，也一直很重要。 这个协议提供了通过化学固定和空气干燥制备贴壁细胞的描述。在这个过程中的重要一步是细胞上的氮化硅窗口，往往不坚持好，使温柔漂洗以特殊的方式成功的关键的增长。

<table><tbody><tr><td>Silicon nitride windows</td><td>Silson Ltd/J B J Business Park/Northampton Rd, Northampton NN7 3DW, United Kingdom</td><td>SiRN-5.0-200-2.0-200. Order by size. Membrane size: 1.5 mm x 1.5 mm. Thickness: 200&amp;nbsp;nm. Frame size: 5&amp;nbsp;mm x 5 mm. Frame thickness: 200 &amp;micro;m.</td><td>Alternate source: SPI Supplies / Structure Probe, Inc. West Chester, PA.</td></tr><tr><td>Reverse tweezers</td><td>Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td><td>78520-5X</td><td>EMS 5X, NC &amp;ndash; Ultra Fine Tweezers</td></tr><tr><td>Rubber grid mat</td><td>Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td><td>71170</td><td>Round Grid Mat</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td><td>338826</td><td>trace metals grade concentrated acetic acid</td></tr><tr><td>PIPES buffer</td><td>Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td><td>P6757</td><td>solid PIPES buffer</td></tr><tr><td>Formaldehyde stock solution</td><td>Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td><td>RT 17113</td><td>10 x 10 ml ampules of 20% aqueous paraformaldehyde</td></tr></tbody></table>

preparing adherent cells for x-ray fluorescence imaging by chemical fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

X射线荧光成像提供关于生物样品的信息的能力，是在被制成以这样的方式即它们是健壮的时间尺度实验辐射损伤这些样品队伍，但它们的化学和结构特征是公保存。在观看已制备如上所述并成像的样品的结果，有可能看到有变化存在的元素-表示定影保留在细胞的这些方面（ 图1）。 相反地​​，在寻找代替在一个样品，其中该处理不顺利，而缓冲保持干燥期间?...

Watch this Scientific Journal Video about Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation at JoVE.com

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

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9.3K 次观看.  Argonne National Laboratory. Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays. 

视频: Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

科研

化学

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

环境科学

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

生物化学

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

生物工程

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

芳香族氨基酸调控细胞代谢的生物正交化学成像

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

生物学

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

通过 3D 相关聚焦离子束铣削制备样品，用于高分辨率冷冻电子断层扫描

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

化学固定准备贴壁细胞的X射线荧光成像

摘要

探索更多视频

此视频中的章节

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

芳香族氨基酸调控细胞代谢的生物正交化学成像

内质网子域的培养细胞中的可视化

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

通过冷冻软X射线断层扫描对细胞进行3D制图描述

通过 3D 相关聚焦离子束铣削制备样品，用于高分辨率冷冻电子断层扫描

化学固定准备贴壁细胞的X射线荧光成像

摘要

探索更多视频

此视频中的章节

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

芳香族氨基酸调控细胞代谢的生物正交化学成像

内质网子域的培养细胞中的可视化

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

通过冷冻软X射线断层扫描对细胞进行3D制图描述

通过 3D 相关聚焦离子束铣削制备样品，用于高分辨率冷冻电子断层扫描

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）