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 border="0" cellpadding="0" cellspacing="0" width="924">
	<tbody>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">silicon nitride windows</td>
			<td style="width:423px;">Silson Ltd/J B J Business Park/Northampton Rd, Northampton NN7 3DW, United Kingdom</td>
			<td style="width:119px;">No part numbers available. Order by size. Membrane size: 1.5 mm x 1.5 mm.&#160; Thickness 500 nm.&#160; Frame size: 5 mm x 5 mm.&#160; Frame thickness: 200 &#181;m</td>
			<td style="width:167px;">Alternate source: SPI Supplies / Structure Probe, Inc.West Chester, PA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">reverse tweezers</td>
			<td style="width:423px;">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 style="width:119px;">78520-5X</td>
			<td style="width:167px;">EMS 5X, NC &#8211; Ultra Fine Tweezers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">rubber grid mat</td>
			<td style="width:423px;">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 style="width:119px;">71170</td>
			<td style="width:167px;">Round Grid Mat</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">acetic acid</td>
			<td style="width:423px;">Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td>
			<td style="width:119px;">338826</td>
			<td style="width:167px;">trace metals grade concentrated acetic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIPES buffer</td>
			<td style="width:423px;">Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td>
			<td style="width:119px;">P6757</td>
			<td style="width:167px;">solid PIPES buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">formaldehyde stock solution</td>
			<td style="width:423px;">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 style="width:119px;">RT 17113</td>
			<td style="width:167px;">10 x 10mL 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 ...

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

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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

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

科研

化学

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学:迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

革兰氏阴性菌的基本金属摄取: x 射线荧光、放射性同位素和细胞分馏

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

软 x 射线吸收光谱和共振非弹性 x 射线散射法检测电池化学成分的元素敏感度

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Chemical Synthesis of Porous Barium Titanate Thin Film and Thermal Stabilization of Ferroelectric Phase by Porosity-Induced Strain

Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its well-balanced ferroelectricity, piezoelectricity, and dielectric constant. In addition, BT does not contain any toxic elements. Therefore, it is considered to be an eco-friendly material, which has attracted considerable interest as a replacement for lead zirconate titanate (PZT). However, bulk BT loses its ferroelectricity at approximately 130 &#176;C, thus, it cannot be used at high temperatures. Because of the growing demand for high-temperature ferroelectric materials, it is important to enhance the thermal stability of ferroelectricity in BT. In previous studies, strain originating from the lattice mismatch at hetero-interfaces has been used. However, the sample preparation in this approach requires complicated and expensive physical processes, which are undesirable for practical applications.
In this study, we propose a chemical synthesis of a porous material as an alternative means of introducing strain. We synthesized a porous BT thin film using a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles were used as an organic template. Through a series of studies, we clarified that the introduction of pores had a similar effect on distorting the BT crystal lattice, to that of a hetero-interface, leading to the enhancement and stabilization of ferroelectricity. Owing to its simplicity and cost effectiveness, this fabrication process has considerable advantages over conventional methods.

Here, we present a protocol for the synthesis of porous barium titanate (BaTiO3) thin film by a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles are used as an organic template.

孔隙诱导应变法合成多孔钛酸钡薄膜及铁电相热稳定性

Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

配体介导成核与钯金属纳米粒子的生长

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

高含量成像分析研究。

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...