部分支持这项工作是由格兰特没有。 213/09从以色列科学基金会，授予没有。 2005258从BSF的基础上，授予没有。 201665从欧洲研究委员会（ERC），由罗素Berrie在Technion工业的纳米技术研究所。我们承认Schulich商学院在Technion工业有关的供应和处理蓝藻的化学教授诺姆ADIR和法里斯萨拉梅的帮助。 Technion工业微纳米加工单位的帮助和支持斯韦特兰娜Yoffis是极大的赞赏。

1。 ESR的显微成像概述首先，我们提供一个简要的解释的ESR，血沉显微镜，和我们的系统的各个组成部分，然后我们将描述实际的成像实验。 电子自旋共振光谱技术中，在特定频率的电磁辐射吸收与未成对电子的自旋的分子，放在下一个外部的静磁场（图1）。 ESR是在广泛领域的科学，如化学，生物，物理，材料科学，聘用，自由基与顺磁中心的检测和鉴定。它是一种顺磁性分子在活体物种的研究环境的强大方法，并提供有关酸度（PH），粘度，氧和活性氧浓度 3的信息。 异构样品，ESR光谱信息，可以得到在空间分辨的方式（即通过获取图像），通过利用磁场梯度 4 。这是非常相似的核磁共振成像（MRI），更常用的方法主要是观察质子的自旋。截至目前为止，如ESR成像技术被应用于几厘米的规模比较大的，在毫米级分辨率的活标本。 （例如，参见图2，参考文献5。）ESR成像较近期的发展寻找小动物在毫米级分辨率毫米波和亚毫米大小的样品测量其功能扩展微米级的分辨率。此字段被称为ESR显微镜，今天可以提供三维ESR的图像接近1微米（参见图3中的有代表性的例子）的决议。 ESR的显微镜是一个传统的ESR光谱仪基本相似。它有一个产生静电场，一个微波系统的自旋激发和信号检测，拿着样品探头，和一个计算机化的控制台来控制采集过程和数据处理的磁铁。其他元件独特的ESR成像和现有的ESR microcopy也梯度磁场源，是电子系统的一部分，和梯度线圈，成像探头位于。我们的特定系统的显示更多的细节在协议中的电影和参考文献2中所述。 2。 ESR显微成像样品制备这一阶段描述的ESR显微成像实验样品制备方法。在这个阶段的细胞结束被放置在一个专门准备的玻璃ESR镜样品容器的底部，连同一个三苯甲基自由基6缓冲液。本协议描述了测量蓝藻细胞，因此，对于其他类型的细胞，适当调整可能需要在样品制备阶段。 <ol><li>首先，几个平方〜400 400微米大小的吸水纸，并插入到Eppendorf管中，随后充满蓝藻悬浮1.2毫升（浓度40毫克/毫升）。 </li><li>停牌是在6000转速在微量离心2分钟。 </li><li>在此之后，上清缓冲区完全删除除外〜50μL左避免蓝藻脱水。作为这一进程的结果，现在是饱和的吸水纸蓝藻细胞。 </li><li>用细镊子，少数纤维是从文件中提取并置于杯专门准备的玻璃样品架7底部。以下3 BG - 11解决方案8，9毫米的三苯（见图1）添加罚款注射器援助样品架。持有人，然后采用紫外光固化胶密封，留下一个小风口开放。 <table border="1"><tbody><tr><td> 库存4 </td><td> 股票3 </td><td> 股票2 </td><td> 库存1 </td></tr><tr><td> H 3 BO 3 2.86g/liter </td><td> K 2 HPO 4：3H 2 O 4.0g/liter </td><td> 硫酸镁 ： 7H 2 O 7.5g/liter </td><td>那2毫克 EDTA 0.1g/liter </td></tr><tr><td> MnCl 2：4H 2 O 1.81g/liter </td><td></td><td></td><td>铁铵柠檬酸0.6g/liter </td></tr><tr><td> 硫酸锌：7H 2 O 0.222g/liter </td><td></td><td></td><td>柠檬酸：1H2O 0.6g/liter </tD&gt; </tr><tr><td> 4：硫酸铜5H 2 O 0.079g/liter </td><td></td><td></td><td>氯化钙2 · 2H 2 O 3.6g/liter </td></tr><tr><td> COCl 2 ：· 6H 2 O 0.050g/liter </td><td></td><td></td><td></td></tr><tr><td> NaMoO 4 · 2H 2 O 0.391g/liter </td><td></td><td></td><td></td></tr></tbody></table> 计划1。BG - 11培养基的制备。 </li></ol> 3。 ESR显微成像实验<ol><li>要开始了成像实验，打开的ESR显微成像系统和谐振器内成像探头插入样品。 </li><li>现在，使用计算机控制软件，设置系统的“调教”模式，并找到共振微波频率的探头，将使用的ESR测量。 </li><li>以下的价值相匹配应用的微波频率，设置静态磁场，脉冲序列的时序参数和观察的ESR信号，以确保系统的功能和样品是精心准备。 </li><li>然后，将成像参数，如像素数，梯度的力量，和其所需的值的梯度脉冲的长度。 </li><li>以下设置，收集interpulse分离，价值观500，600，700 NS一个哈恩回波成像脉冲序列（图4）三个ESR的3D图像。 </li><li>样品投射灯是根据所需的实验条件下打开或关闭。 </li><li>在收购过程中，数据将自动保存。这些原始数据文件，然后通过Matlab软件脚本提供的三苯甲基自由基浓度和弛豫时间T 2的地图，这是翻译成通过现有的预校准的氧气浓度形象的图像处理。 </li></ol> 4。代表性的成果实验的结果是几个记录在不同的τ值的三维ESR微图像。图5典型的原始数据图像。前三名的图像，在黑暗条件下测得，除了在减少信号强度非常相似。另一方面，光线照射下的图像格局的变化，由于在不同部位的样品的不同的弛豫时间。此数据可处理1获得振幅图像，如图6所示和弛豫时间 T 2（图7）的图像。 T 2图像转化为通过一个预先存在的校准曲线，链接通过方程的弛豫时间的氧气浓度的氧浓度值： <img src="/files/ftp_upload/2122/2122eq.jpg" alt="公式1" />在这里，T 2 0探头的自旋弛豫时间在缺氧条件下（取决于探头的浓度，C和它的扩散系数D），k是比例常数。在大多数情况下，扩散系数不变化为生活样本（虽然，如果需要的话，它可以在原则上可以直接评估也由ESR 6，10），并在成像过程中获得的自旋浓度。因此，这种关系可用于直接测量氧气浓度。 回到图6，它是显而易见的，从幅度图像，蓝藻的细胞主要位于右侧的样品架。此外，根据图7，它是明确的，光启动生产的O 2，导致在该解决方案的O 2浓度显着增加，主要是在附近的蓝藻的体素。 <img src="/files/ftp_upload/2122/2122fig1.jpg" alt="图1" /> 图1：电子自旋共振的能量水平。 <img src="/files/ftp_upload/2122/2122fig2.jpg" alt="图2" /> 图2：典型的氧气浓度对荷瘤鼠的形象。左边的图像显示的解剖信息，根据MRI图像。一个稳定的有机自由基注入鼠标和ESR特性提供了在其环境中的氧气浓度（右）。 ESR为基础的结果MRI解剖图像上叠加。视场是32毫米。 <img src="/files/ftp_upload/2122/2122fig3.jpg" alt="图3" /> 图3：高分辨率的微观尺度ES的两个例子ř图像的光刻生成N @ C 60的粉末（左）和LIPC顺磁性晶体样品（右） <img src="/files/ftp_upload/2122/2122fig4.jpg" alt="图4" /> 图4：典型的哈恩成像脉冲序列显示微波（MW）和梯度，G X，G y和g ž脉冲。 <img src="/files/ftp_upload/2122/2122fig5.jpg" alt="图5" /> 图5：典型的原始数据ESR的微观图像：A，B和C为τ= 500600700 NS，分别测量没有光照的蓝藻样品的原始数据。项目D，E，并为A，B和C，但光照相同。强度是绘制在任意规模（但在每3个或深或浅的原始数据图像的设置一致） <img src="/files/ftp_upload/2122/2122fig6.jpg" alt="图6" /> 图6：相应的解决方案中的自由基浓度（任意缩放）幅图像。 <img src="/files/ftp_upload/2122/2122fig7.jpg" alt="图7" /> 图7：T 2图像和相应的[O 2]暗（左）和光（右）的条件下的值。

<ol>
	<li>Halevy, R., Tormyshev, V., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-imaging+of+Oxygen+Concentration+near+Live+Photosynthetic+Cells+by+Electron+Spin+Resonance.">Micro-imaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance.</a> Biophysical Journal. , (2010).</li><li>Blank, A., Suhovoy, E., Halevy, R., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESR+imaging+in+solid+phase+down+to+sub-micron+resolution:+methodology+and+applications.">ESR imaging in solid phase down to sub-micron resolution: methodology and applications.</a> J Phys Chem. 11, 6689-6699 (2009).</li><li>Gallez, B., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+EPR:+when,+how+and+why?.">In vivo EPR: when, how and why?.</a> NMR Biomed. 17, 223-225 (2004).</li><li>Eaton, G. R., Eaton, S. S., Ohno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EPR imaging and in vivo EPR. , (1991).</li><li>Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+tumor+oxygenation+and+microvascular+permeability+using+Overhauser+enhanced+MRI.">Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.</a> Proc Natl Acad Sci USA. 106, 17898-17903 (2009).</li><li>Talmon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Porous+Media+by+PGSE.">Molecular Diffusion in Porous Media by PGSE.</a> ESR. J Phys Chem. , (2010).</li><li>Halevy, R., Talmon, Y., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+production+of+glass+sample+holders+for+improved+sensitivity+and+resolution+in+ESR+microscopy.">Photolithographic production of glass sample holders for improved sensitivity and resolution in ESR microscopy.</a> Applied Magnetic Resonance. 31, 591-598 (2007).</li><li>Allen, M. M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+Division+of+Some+Unicellular+Blue-Green+Algae.">Growth and Division of Some Unicellular Blue-Green Algae.</a> J Gen Microbiol. 51, 199-199 (1968).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> J Gen Microbiol. 111, 1-61 (1979).</li><li>Blank, A., Talmon, Y., Shklyar, M., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+diffusion+in+liquid+phase+by+electron+spin+resonance.">Direct measurement of diffusion in liquid phase by electron spin resonance.</a> Chem Phys Lett. 465, 147-152 (2008).</li></ol>

此协议表明如何ESR的显微成像可以应用到地图氧浓度附近居住的小样本。一个空间分辨率〜30 × 30 × 100微米证明，与附近的微摩尔的氧气浓度的敏感性和分飞摩尔每个体素的绝对氧敏感性。氧细胞附近的映射使用的ESR显微成像补充基于微电极或荧光/磷光目前可用的技术。此外，适当的顺磁探头，它会很容易地适用于细胞内的氧气显微成像，能力等方法发现很难实现。在不久的将来，我们计划进一步改进这种方法，提供活的样本图像，几微米的分辨率，提供对比参数，如超氧化物浓度，酸度（PH），探头的扩散系数，当然，氧气浓度。这些功能是相辅相成的对比类型和样本特征（非透明如厚样品，并在某些情况下，细胞内与外的测量）到目前的光学方法。

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Centrifuge </td>
			<td>&nbsp;</td>
			<td>Kendro</td>
			<td>Heraus, 75003235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Perdeuterated triarylmethyl (trityl) radical</td>
			<td>&nbsp;</td>
			<td>Synthesized at Novosibirsk using the method described in reference 6.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BG-11 buffer</td>
			<td>&nbsp;</td>
			<td>For instruction preparation, see Scheme 1 and references 8, 9. </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>&nbsp;</td>
			<td>Hamilton</td>
			<td>Microliter 7000.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultraviolet Curing</td>
			<td>&nbsp;</td>
			<td>Norland Products, Inc.</td>
			<td>NOA63, or NOA61.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

electron spin resonance micro-imaging of live species for oxygen mapping

这个协议描述三维映射与微米级的分辨率1活细胞的直接环境中的氧含量的电子自旋共振（ESR）显微成像方法。氧气是在生命周期中最重要的分子之一。它作为在线粒体氧化磷酸化的终端电子受体和用于生产活性氧。氧气测量是很重要的线粒体和代谢功能，信号转导通路，各种刺激的影响，细胞膜的通透性，和疾病的分化的研究。耗氧量，因此，细胞的新陈代谢，这是广泛适用于各种生物系统对整个生物体的细胞线粒体的信息标记。由于其重要性，有很多方法被开发为氧气的测量实时系统。主要是根据当前的尝试，提供高分辨率的氧成像光学荧光和磷光方法，未能提供令人满意的结果，因为他们采用高光毒性和含氧量低灵敏度的探测器。血沉增快，措施，从外源性样品中顺磁探头的信号，被称为氧气的浓度，以提供非常精确的测量。在一个典型的案例，ESR测量地图探头的线型扩大和/或松弛时间缩短，直接链接到当地的氧气浓度。 （氧是顺磁性，因此，外生顺磁探头碰撞时，急促的弛豫时间。）传统上，这些类型的实验与分辨率低，毫米波规模小动物成像的ESR。在这里，我们将展示如何ESR成像还可以进行微米级的小活标本的检查。 ESR的显微成像是一个相对较新的方法，使收购接近室温 2 1微米的分辨率与空间分辨ESR信号。本议定书的文件的主要目的是为了表明，这种新方法，随着新开发的氧敏感探头，可以适用于小活样品中的氧含量的映射。一个空间分辨率〜30 × 30 × 100微米证明，与附近的微摩尔的氧气浓度的敏感性和分飞摩尔每个体素的绝对氧敏感性。氧细胞附近的映射使用的ESR显微成像补充基于微电极或荧光/磷光目前可用的技术。此外，适当的顺磁探头，它也将很容易地适用于细胞内的氧气显微成像，能力等方法发现很难实现。

Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

这个协议描述为微米级的三维成像在活细胞的电子自旋共振显微镜直接环境氧浓度的方法。

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

electron-spin-resonance-micro-imaging-live-species-for-oxygen

Research

JoVE Journal

Biology

22.2K 次观看.  The Technion, Israel Institute of Technology. 这个协议描述为微米级的三维成像在活细胞的电子自旋共振显微镜直接环境氧浓度的方法。

视频: Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

科研

生物学

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

通过可切换原卟啉IX依赖性原位活性氧物种光生成刺激小鼠皮肤中的干细胞壁龛和组织再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

评估交配蜜蜂女王的农用化学品风险

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...