来自美国国家卫生研究院的资助EB007279和HL081273的支持表示感谢。

1。氧气测量协议的一般描述 （本节不会有任何行动，但对于理解本文的其余部分的关键。例如，它可以被拍摄下来，，作为一个序列数的Power Point幻灯片，伴随着的声音。） 1.1）探头传递到利益的媒介，例如，在一个动物的血液或组织间液注射。 1.2）的对象（组织表面）是适当波长的光照亮，以促进其激发三重态的探测。通常情况下，激发发生通过单光子的机制。然而，在特殊的双光子增强探头的情况下，激发可以通过双光子的机制，它允许使用高分辨率显微镜应用的探头。单光子激发通常会提供较少的空间分辨率，但需要简单的仪器，可用于在与基于LED光纤phosphorometers的单点测量。 1.3）由探头发出的磷光来源于长寿命的三重态。必须专门设计的探针分子，给予高度的三重态量子产率，并发出荧光磷光。在三重态的同时，探头可以体验与氧分子，它可以激活三重态的碰撞交锋 - “淬火”过程被称为在没有氧气，三重态的寿命，和磷光衰减，因此，τ 0。在氧气存在的磷光寿命（τ）是缩短淬火。之间的三重态寿命和环境中的氧气量有直接的关系。这是知名的Stern - Volmer关系，它连接的磷光寿命（τ），氧气浓度[O 2]（或氧分压PO 2）： <img src="/files/ftp_upload/1731/1731equation.jpg" alt="方程" />在生物系统中，氧气是迄今为止最有效的淬灭的磷光，这使得非常有选择性的对氧气的磷光猝灭法。 1.4）为了衡量寿命吨，分级发出的磷光光子的时间。例如，激励脉冲后的，200光子可能被收集在第15微秒，在未来15微秒收集150光子，等等，直到没有光子被收集。在回收箱的数目绘制与时间的磷光衰减，这是分析产量的寿命τ。成像，在此过程适用于图像的每个像素，在磷光寿命地图。寿命测量的对象，这是常见的生物样本的探针整个分布的非均质性不敏感。因此，报告只对氧的寿命。寿命τ转换成氧气浓度使用的Stern - Volmer关系。 2。磷光探针的建设磷光探针在生物系统中的氧气测量磷光生色（核心），封装成的树枝状构造的笼子，包括 - 高经常超支化聚合物[3]。树枝状从与环境的相互作用保护的核心和氧的扩散率控制的核心，使我们能够优化探头的灵敏度和动态范围（常数k的Stern - Volmer关系的 Q）。树枝状总站修改与化学惰性的亲水性的聚乙二醇（PEG）群体，使探针高度水溶性，并​​防止其与生物大分子的相互作用。 磷光探针核铂或钯卟啉和π扩展卟啉配合物。 π扩展卟啉的吡咯环卟啉与外部的芳香片段融合。 π扩展允许探头光谱性质的调整，以满足一个特定的成像应用程序（如显微镜与断层扫描）的要求。作为一个例子，我们描述的合成钯tetaaryltetrabenzoporphyrins（PdAr TBP）[4]。 PdAr 4 TBP的强大的吸收带在远红外频谱和强烈的磷光的一部分，非常适合生物耗氧量测。 PdAr 4磷酸三丁酯的合成包括以下步骤： 2.1）的制备tetraaryltetracyclohexenoporphyrins林德塞凝结（Ar4TCHP）[5]与取代苯甲醛tetrahydroisoindole。以0.01 M解决方案的tetrahydroisoindole在CH（1 EQ）&lt;分&gt; 2 CL 2的芳香醛（EQ）是加入氩气保护下。反应混合物在室温下BF 3 XET 2 O（0.2当量）搅拌10分钟，在黑暗的补充，并增加2小时，反应混合物在室温下搅拌DDQ的（1 EQ）增加了，一夜之间只剩下连续搅拌和混合。解决的办法是冲10％AQ NA 2 SO 3，10％AQ NA 2 CO 3，5％水溶液HCL，最后用盐水。 NA 2 SO 4干燥有机相，然后在真空浓缩。残渣再结晶CH 2 CL 2 - 叔丁基醚的混合物，给AR 4 TCHP绿色粉末。产量约50％。 2.2）钯插入获得PdAr 4 TCHPs。自由基卟啉（EQ）与PdCl 2或Pd（OAC）2（1.2当量）超过3 N ET（20式）的存在回流CH 3 CN治疗。转换监测紫外可见光谱（溶剂CH 2 CL 2，醋酸），并认为完成后，在468-472 nm的Soret带听写消失。混合物被允许降温，稀释CH 2 Cl 2等 ，并透过一层薄薄的硅藻土过滤除去钯（0） 。溶剂蒸干，残渣是由硅胶作为溶剂使用CH 2 CL 2列色谱纯化。暗红色的部分是收集，溶剂蒸发给PdAr 4 TCHPs产量几乎定量目标。 2.3）PdAr 4 TBP的准备PdAr 4 TCHPs氧化与DDQ的2倍过量回流的四氢呋喃（16式） 。在回流过程中，颜色从暗红色到深绿色。溶剂蒸干，残渣稀释与CH 2 CL 2，与10％AQ NA 2 SO 3，水和盐水洗涤。有机相NA 2 SO 4干燥和真空浓缩。残余物经硅胶层析（洗脱液CH 2 CL 2）。第一个暗绿色的一小部分是收集，集中在真空中给予85-90％PdAr4TBP的蓝绿色粉末。 2.4）钯周边酯基四 - （3,5 - dibutoxycarbonylphenyl）tetrabenzoporphyrin（PdAr4TBP）使用氢氧化钾的10倍超过1％（V / V）在室温下的水/四氢呋喃水解。溶剂，倒出PdAr4TBP的羧酸衍生物的形成钾盐沉淀。固体溶解于水，搅拌2小时浓度酸化，。盐酸pH〜4-5的。获得的沉淀，通过离心分离，用清水洗净，真空干燥。 保护树状分子 ，每个树枝状的个别分支称为dendron。保护树状分子构造芳基甘氨酸（AG）的积木。公司树状分子可以很容易地从便宜的起始原料合成和高纯度和色谱方法的收益率隔离。第2代（G2），AG - dendron的合成包括以下步骤： 2.4）的合成，积木，BOC保护的3,5 - dicarboxyphenyl glycineamide和3,5 - dibutoxycarbonylphenyl glycineamide，菲舍尔haloacyl卤化物方法使用。 2.5）建设CDMT / NMM肽耦合化学块耦合。 2.6）Deprotecting搅拌，在室温下为2 h的G2在TFA的dendron解决方案的重点氨基酸组 2.7）提出了更高的一代树状分子组装，耦合/去保护步骤是重复的。 树枝状大分子组装是通过使用HBTU / DIPEA耦合化学deprotected树状分子耦合PdAr4TBP（或其他卟啉）。 2.8）首先，从dendron删除叔丁氧羰基保护的群体。例如，BOC保护G3 dendron（0.58克），溶解在TFA（15毫升），和左混合物在室温下反应2h后。酸被删除，旋转蒸发，产生TFA的盐dendron。残留物溶解在NMP的干（10毫升），并分别加入几滴DIPEA，解渴的TFA的痕迹。 2.9）开始之前偶联反应，关键是完全溶解的卟啉。例如，PT tetracarboxyphenylporphyrin（0.061克）下加热溶解干NMP（65毫升）140 ° C为10分钟，氮气流量下，。 2.10）溶液冷却到室温，HBTU（0.211克）补充说，混合，搅拌5分钟。 2.11）DIPEA（0.35毫升）加入到混合物中的一个部分，后面紧跟增加soluti对反式脂肪酸盐dendron在NMP（见2.8），和混合留给下搅拌过夜。 2.12）的混合物倒入3％AQ。氯化钠（350毫升），和由此产生的沉淀，离心收集，并与水，甲醇， 等 2洗净，重复暂停/离心产量目标卟啉-树枝状。 2.13），水解的外围酯基的树枝状NME 4 OH（〜5毫米）在DMSO /甲醇20-60分钟内完整的溶剂去除，先用。此过程使我们能够产生足够数量的羧基水溶性树枝状。要完成水解，树枝状治疗用0.1N氢氧化钠水溶液（过夜）。因此，纯粹的卟啉 - 树枝状羧酸可以隔离在80-95％的产量。 改性树枝状外围 ，在这一点上 。，开始从相同的卟啉-树枝状，无论是一个或两个光子探头可以合成。在第一种情况下，所有外围的羧基修改与聚乙二醇单位-聚乙二醇作为已知的方法。在后一种情况下，几个羧基加上双光子天线的生色（香豆素343）修改与乙二胺（EDA）连接器，其余的羧基的聚乙二醇化[6]。树枝状被使用monomethoxyoligoethyleneglycolamine（M - PEG - NH 2，音像兆瓦1000）聚乙二醇采用HBTU / DIPEA耦合化学。 2.14）dendron NMP（或DMF）（1-10毫米）的1.25倍HBTU过剩的解决方案增加了，反应混合物在室温下搅拌10分钟。增加了6.25倍DIPEA超过，随后立即此外M - PEG - NH2的1.25倍超额。 2天在室温下搅拌反应混合物。乙醚被添加到反应混合物中分离，形成沉淀从四氢呋喃乙醚此外离心和重新沉淀。 2.15）最终纯化是实现与前第一阶段收集体积排阻色谱。卟啉 - 树枝状聚乙二醇溶解于少量的THF（约15毫升），装上聚苯乙烯美国证券交易委员会阶段（BIORAD，Biobeads的S - X1）填补了色谱柱的顶部。列纯四氢呋喃洗脱，收集和深色乐队前。 2.16）四氢呋喃旋转蒸发器上被删除，剩余材料真空干燥。 3。探头表征光物理特性包括测量探头的吸收和发射光谱，磷光寿命τ0和氧猝灭的Stern - Volmer常数k q下的生理状况。 3.1）的吸收和发射光谱的环境条件下获得的使用标准仪器（分光光度计和稳定的荧光）。 〜1探头微米的解决方案是使用。 3.2）接下来，获得的Stern - Volmer校准情节。这是最重要的测量，因为它使我们能够与探头的寿命的氧气浓度。 3.3）探头在PBS缓冲液（pH值7.2）的解决方案是放置在一个特殊的圆柱形比色皿，这里面的温度控制腔定位的激发和发射光纤端口，位于内光防渗笼。纤维被带进密切接触的比色皿内直角几何室。 3.4）试管关闭一个塞子，在其中插入一个高度敏感的Clark型氧电极（CK -氧电极）。瓶塞也包含两个氩气的进气口和插座的针端口。电极浸入到该解决方案，而仅仅提供上述溶液的表面流动的气体流量针。起初，氩气是不连接的入口。 3.5）的温度设定为所需的值（通常为36-37℃）和解决方案是平衡搅拌下离开。 3.6）的激发光纤连接到数字phosphorometer激发端口，通过PC操作。在phosphorometer光源是一个大功率LED，其输出是333 kHz的D / A板控制。排放光纤连接到另一个phosphorometer，这是一个高度敏感的红外APD（雪崩光电二极管）耦合到的光学端口。二极管的输出放大到相同的控制电路板，它允许的激发和发射通道之间的同步的A / D通道美联储。 3.7）控制软件人任何所需的长度（如5或10毫秒），收集在任意选择的时期（一般为2-3毫秒）的磷光衰减，激发脉冲的低点一代。一个单一的寿命测量所需的时间通常为0.5-1小号，但是，测量可以得到迅速10-20每秒。 3.8）氧电极的输出被放大到另一个A / D板的指示同一台PC上。这是一种低频率的板（1 kHz最大），它是用来记录在选定的时间段电极的电流。记录电极的数据的程序与phosphorometer软件同时运行。 3.9），一旦溶液的温度平衡，phosphorometer和电极计划初始化在同一时间进行测量，每10秒其输出成两个单独的文件同步记录。之后，氩气的试管塞的进气口连接到。 3.10）由于氩搅拌溶液的表面流动的，它逐渐取代了氧气。在一个电极的电流减少和增加的磷光寿命，这是phosphorometer测量结果。通常情况下，氧气是流离失所形成约2 h后完全解决方案，在这段时间内的数据完全自动记录到文件中。 3.11）滴定运行结束后，电极的数据和磷光寿命导入到一个标准的分析程序（如Miocrocal原产）。电极的电流线性依赖于氧气的浓度，电极使用它几乎是零零氧。在大气压力下，PO 2是已知的（约150毫米汞柱）。因此，电极的数据可以直接转换为氧气的规模，并与PO 2逆磷光寿命曲线可以构造。此图是安装与使用最小二乘法直线线，其斜率恒定KQ给予氧气淬火。磷光寿命T0是，无论是从同一个适合（截距的倒数），或直接从零氧测量。 3.12），如果需要，滴定重复使用白蛋白的存在探头解决方案 - 目前，在高浓度的血浆中的一种蛋白质 - 为了模拟条件满足在现实实验系统（一种动物的血液在体内 ）。应该是相同的，如果树枝状探头保护和隔离接触与白蛋白的PEG组探头获得的Stern - Volmer图。否则，探头和血液中的蛋白质相互作用，并，这将导致改变的Stern - Volmer情节，造成氧气测量的模糊性。 由此获得的校准常数使用中的氧气浓度是一个先验未知的成像实验。

<ol>
	<li>Vanderkooi, J. M., Maniara, G., Green, T. J., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+method+for+measurement+of+dioxygen+concentration+based+on+quenching+of+phosphorescence.">An optical method for measurement of dioxygen concentration based on quenching of phosphorescence.</a> J. Biol. Chem. 262, 5476-5482 (1987).</li><li>Rumsey, W. L., Vanderkooi, J. M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+phosphorescence:+A+novel+method+for+measuring+the+distribution+of+oxygen+in+perfused+tissue.">Imaging of phosphorescence: A novel method for measuring the distribution of oxygen in perfused tissue.</a> Science. 241, 1649-1651 (1988).</li><li>Lebedev, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+phosphorescent+probes+for+oxygen+Imaging+in+biological+systems.">Dendritic phosphorescent probes for oxygen Imaging in biological systems.</a> Acs Applied Materials and Interfaces. 1, 1292-1304 (2009).</li><li>Finikova, O. S., Cheprakov, A. V., Beletskaya, I. P., Carroll, P. J., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+versatile+synthesis+of+substituted+tetrabenzoporphyrins.">Novel versatile synthesis of substituted tetrabenzoporphyrins.</a> Journal of Organic Chemistry. 69, 522-535 (2004).</li><li>Lindsey, J. S., Schreiman, I. C., Hsu, H. C., Kearney, P. C., Marguerettaz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rothemund+and+Adler-Longo+Reactions+revisited:+Synthesis+of+tetraphenylporphyrins+under+equilibrium+conditions.">Rothemund and Adler-Longo Reactions revisited: Synthesis of tetraphenylporphyrins under equilibrium conditions.</a> Journal of Organic Chemistry. 52, 827-836 (1987).</li><li>Lebedev, A. Y., Troxler, T., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+metalloporphyrin-based+dendritic+nanoprobes+for+two-photon+microscopy+of+oxygen.">Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen.</a> J. Porphyrins and Phthalocyanines. 12, 1261-1269 (2008).</li></ol>

<table border="1" cellspacing="0" cellpadding="0"&gt;<tr<td<strong&gt;缩写</strong<strong</strong</td&gt;<td<strong&gt;姓名</strong<strong</strong</td&gt;</tr&gt;<tr<td&gt; NMP</td&gt;<td&gt; N - methylpyrrolidinone</td&gt;</tr&gt;<tr<td&gt;反式脂肪酸</td&gt;<td&gt;三氟乙酸</td&gt;</tr&gt;<tr<td&gt; DIPEA</td&gt;<td&gt;二异丙基</td&gt;</tr&gt;<tr<td&gt; HBTU</td&gt;<td&gt; 2 - （1H -苯并三唑-1 - 基）-1,1,3,3 - tetramethyluronium六氟磷酸</td&gt;</tr&gt;<tr<td&gt;二甲基亚砜</td&gt;<td&gt;二甲基亚砜</td&gt;</tr&gt;<tr<td&gt; CDMT</td&gt;<td&gt; CDMT = 1 - 氯-3,5 - dimethoxytriazine</td&gt;</tr&gt;<tr<td&gt; NMM</td&gt;<td&gt; NMM = N - methylmorfoline</td&gt;</tr&gt;</table

synthesis and calibration of phosphorescent nanoprobes for oxygen imaging in biological systems

氧气测量磷光猝灭[1，2]包括以下步骤：1）探头被交付到利益的媒介（如血液或组织间液）; 2）对象是适当波长的光照射，以激发探测到它的三重态; 3）所发出的磷光是收集，分析其时间当然是产生的磷光寿命，这是转换成氧气的浓度（或分压，PO<sub&gt; 2</sub&gt;）。探头必须与生物环境相互作用，并在某些情况下4）从媒介测量完成后排出体外。上述每个步骤规定上的磷光探针，从而构成侵入组件的唯一的测量协议的分子设计的要求。在这里，我们审查为在生物系统中的氧气测量的树突状磷光纳米传感器的设计。探头由铂或钯卟啉的polyarylglycine（AG）树枝状，改性聚乙二醇（PEG）残留外围。对于有效的双光子激发，总站的树枝状双光子天线生色，捕获的激发能量通过分子内和通道探头的三重核心的FRET（福斯特共振能量转移）可能会被修改。我们描述的光物理性质的探头和目前详细的校正协议的关键。

Watch this Scientific Journal Video about Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems at JoVE.com

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

我们目前磷光淬火和审查设计基于卟啉 - 树突状纳米传感器在生物系统中的氧气成像的氧气测量的原则。

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

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13.8K 次观看.  University of Pennsylvania. 我们目前磷光淬火和审查设计基于卟啉 - 树突状纳米传感器在生物系统中的氧气成像的氧气测量的原则。

视频: Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

ACT-PRESTO：生物组织清算和体积成像方法免疫标记

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.