Name: measurement of total calcium in neurons by electron probe x-ray microanalysis
Uploaded: 2013-11-20T17:30:00
Description: Watch this Scientific Journal Video about Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis at JoVE.com

我们要感谢恭答温特斯女士为优秀的技术援助。这项工作是由NINDS院内研究计划，美国国立卫生研究院（Z01 NS002610）的基础神经科学计划的支持。

这里介绍的方法是使用专用仪器，工具和软件开发。因为实验室将不会使用相同的实验装置，该方法是广义的，其中可能的。 1。快速凝固将要描述的分析方法是绝对依赖于低温的方法为：细胞或组织中的1）中的“冷冻固定”的方式，定量保留扩散性的组织成分和化学元素的分布，因为他们是在活细胞在冷冻的瞬间和2）极薄的制备冷冻切片适于成像和分?...

<ol>
	<li>Aronova, M. A., Kim, Y. C., Pivovarova, N. B., Andrews, S. B., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+EFTEM+mapping+of+near+physiological+calcium+concentrations+in+biological+specimens.">Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens.</a> Ultramicroscopy. 109, 201-212 (2009).</li><li>Aronova, M. A., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+mapping+by+electron+energy+loss+spectroscopy+in+biology.">Elemental mapping by electron energy loss spectroscopy in biology.</a> Methods Mol. Biol. 950, 209-226 (2013).</li><li>Bezprozvanny, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+neurodegenerative+diseases.">Calcium signaling and neurodegenerative diseases.</a> Trends Mol. Med. 15, 89-100 (2009).</li><li>Fernandez-Segura, E., Warley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+probe+X-ray+microanalysis+for+the+study+of+cell+physiology.">Electron probe X-ray microanalysis for the study of cell physiology.</a> Methods Cell Biol. 88, 19-43 (2008).</li><li>Gibson, G. E., Starkov, A., Blass, J. P., Ratan, R. R., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+consequence:+Mitochondrial+dysfunction+initiates+and+propagates+neuronal+dysfunction,+neuronal+death+and+behavioral+abnormalities+in+age-associated+neurodegenerative+diseases.">Cause and consequence: Mitochondrial dysfunction initiates and propagates neuronal dysfunction, neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases.</a> Biochim. Biophys. Acta. 1802, 122-134 (2010).</li><li>Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+in+electron+microscopy.">Novel techniques in electron microscopy.</a> Curr. Opin. Neurobiol. 14, 591-598 (2004).</li><li>LeFurgey, A., Bond, M., Ingram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontiers+in+electron+probe+microanalysis:+application+to+cell+physiology.">Frontiers in electron probe microanalysis: application to cell physiology.</a> Ultramicroscopy. 24, 185-219 (1988).</li><li>Newbury, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+X-ray+mapping:+X-ray+spectrum+imaging+above+100+kHz+output+count+rate+with+the+silicon+drift+detector.">The new X-ray mapping: X-ray spectrum imaging above 100 kHz output count rate with the silicon drift detector.</a> Microsc. Microanal. 12, 26-35 (2006).</li><li>Pierson, J., Vos, M., McIntosh, J. R., Peters, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+electron+cryotomography+of+vitreous+cryo-sections.">Perspectives on electron cryotomography of vitreous cryo-sections.</a> J. Electron Microsc. 60, S93-S100 (2011).</li><li>Pivovarova, N. B., Hongpaisan, J., Andrews, S. B., Friel, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-induced+mitochondrial+Ca+accumulation+in+sympathetic+neurons:+spatial+and+temporal+characteristics.">Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.</a> J. Neurosci. 19, 6372-6384 (1999).</li><li>Pivovarova, N. B., Nguyen, H. V., Winters, C. A., Brantner, C. A., Smith, C. L., Andrews, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+calcium+overload+in+a+subpopulation+of+mitochondria+triggers+delayed+death+in+hippocampal+neurons.">Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.</a> J. Neurosci. 24, 5611-5622 (2004).</li><li>Ritchie, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+simulation+in+DTSA-II.">Spectrum simulation in DTSA-II.</a> Microsc. Microanal. 15, 454-468 (2009).</li><li>Stanika, R. I., Winters, C. A., Pivovarova, N. B., Andrews, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+NMDA+receptor-dependent+calcium+loading+and+mitochondrial+dysfunction+in+CA1+vs.+CA3+hippocampal+neurons.">Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons.</a> Neurobiol. Dis. 37, 403-411 (2010).</li><li>Zhang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+receptor+arrays+in+frozen-hydrated+sections+and+plunge-frozen+specimens+of+E.+coli+engineered+to+overproduce+the+hemotaxis+receptor+Tsr.">Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the hemotaxis receptor Tsr.</a> J. Microsc. 216, 76-83 (2004).</li></ol>

这里介绍的电子显微镜为基础的分析方法允许用于检测，鉴定和生物学意义的一些元素，包括钠，钾，磷，特别是钙的定量。这些分析，可以进行在细胞内， 即内细胞器，由于定位和识别在从快速冷冻标本制备冷冻切片的高品质的图像感兴趣结构的能力的分辨率。注意，没有染色需在结构质量对常规固定，塑料包埋制剂记录可比电子图像，即使在组织元素的位置，定量保留。 <p class="jov...

钙离子可以说是生物学中最重要和最多才多艺的细胞信号转导的实体，打在正常过程中的重要作用等不同的突触传递和基因表达。在另一方面，钙​​是在细胞死亡同样重要。特别是，钙放松管制是在行程的一个关键因素在神经元损伤，帕金森氏症，阿尔茨海默氏病和其它神经变性疾病3,5。因此，这是极其重要的，定量了解如何钙的细胞内分布，以及该改变下列生理或病理生理的刺激。在溶液中游离或结合到基体 - - 这细胞的钙浓度超过几个数量级为刺激的结果改变这个目标是由事实，钙是动态两个物理状态之间分布复杂。 虽然有可供fr的分析几个先进的方法ee值细胞内钙，总钙浓度定义胞内区室中的确定是切实限于一种方法，即，电子探针显微分析（EPMA）。 EPMA是一种技术，伴侣的X-射线光谱仪的透射电子显微镜（TEM）。透射电子显微镜的电子枪聚焦一个静止的，亚微米电子探针的利益和作为发射电子轰击而产生的元素特有的X射线的亚细胞区域收集和分析（见参考文献7,4的详细技术审查）。 EPMA的优点包括单细胞器级的分辨率和submillimolar灵敏度。然而在实践中，EPMA需要专门cryotechniques和仪器对样品的制备和分析。在这里，工具，技术和适用于使用电子探针细胞内钙离子的测量仪器进行了描述。线粒体内钙的特殊Interest是考虑的关键作用，在神经退行性疾病线粒体钙超载的戏剧。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS/MATERIALS</td>
		</tr>
		<tr>
			<td>Thermanox plastic coverslips</td>
			<td>Thermo Fischer Scientific</td>
			<td>72280</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture inserts</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>For 6-well plates</td>
		</tr>
		<tr>
			<td>Cryopins</td>
			<td>Leica Microsystems</td>
			<td>16701952</td>
			<td>Grooved</td>
		</tr>
		<tr>
			<td>Wood applicators</td>
			<td>EM Sciences</td>
			<td>72300</td>
			<td></td>
		</tr>
		<tr>
			<td>Folding EM grids</td>
			<td>Ted Pella</td>
			<td>4GC100/100</td>
			<td>100 mesh</td>
		</tr>
		<tr>
			<td>Indium foil</td>
			<td>Alfa Aesar</td>
			<td>13982</td>
			<td>0.25 mm thick</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Plunge freezing device</td>
			<td>Leica Microsystems</td>
			<td>KF-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Slam freezing device</td>
			<td>LifeCell</td>
			<td>CF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems</td>
			<td>UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoattachment for microtome</td>
			<td>Leica Microsystems</td>
			<td>FC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond cryotrimming tool</td>
			<td>Diatome</td>
			<td>Cryotrim 45</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond cryoknife</td>
			<td>Diatome</td>
			<td>Cryo 35</td>
			<td></td>
		</tr>
		<tr>
			<td>Antistatic device</td>
			<td>Diatome</td>
			<td>Hauf Static Line</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo electron microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>EM912 Omega</td>
			<td></td>
		</tr>
		<tr>
			<td>EM cryo specimen holder</td>
			<td>Gatan</td>
			<td>CT3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-scan CCD camera, 2k x 2k</td>
			<td>Troendle (TRS)</td>
			<td>Sharpeye</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Olympus SIS</td>
			<td>iTEM suite</td>
			<td></td>
		</tr>
		<tr>
			<td>ED x-ray detector</td>
			<td>Oxford Instruments</td>
			<td>Linksystem Pentafet</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Processor</td>
			<td>Oxford Instruments</td>
			<td>XP-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PCI backplane card</td>
			<td>4pi Systems</td>
			<td>Spectral Engine II</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Apple</td>
			<td>Any OS9-compatible model</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray analysis software</td>
			<td>NIST</td>
			<td>DTSA, DTSA II</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet software</td>
			<td>Microsoft</td>
			<td>Excel</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<ol>
				<li>The CF100 is no longer sold commercially, although the machine is available at many academic facilities, and complete machines or parts can be found on-line.</li>
				<li>A video tutorial for the CT3500 cryotransfer holder is available at <a href="http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4" target="_blank">http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4</a>.</li>
				<li>The SEII is obsolete; the Universal Spectral Engine Is a later, PC-compatible product with comparable functionality. 4pi has ceased manufacturing and sales but still provides technical customer support. Used systems are often found online.</li>
				<li>The original DTSA is now obsolete. NIST offers in the public domain an updated successor, DTSA II 12 (<a href="http://www.nist.gov/mml/mmsd/software.cfm" target="_blank">http://www.nist.gov/mml/mmsd/software.cfm</a>)</li>
			</ol>
			</td>
		</tr>
	</tbody>
</table>

measurement of total calcium in neurons by electron probe x-ray microanalysis

在这篇文章中使用被称为电子探针（EPMA）技术的工具，手段，以及适当的细胞内元素含量的定量测量工具的描述。线粒体内钙是因为线粒体钙超载起着神经退行性疾病中的关键作用特别关注。该方法是基于X射线的分析电子显微镜（EM）由与试样的电子束的相互作用而产生的。为了保持在电子显微镜标本的扩散性元素的天然分布，EPMA需要组织随后的超薄冷冻切片的制备“冷冻固定”。培养的细胞或器官切片文化的快速冷冻是通过急跌液态乙烷或满贯冷冻结冰对寒冷的金属块，分别。名义上冰冻切片80 nm厚的是在约切干用钻石刀。 -16076，C，安装在碳/聚乙烯醇缩醛涂层的铜网上，并cryotransferred成冷冻电镜使用的是专门的cryospecimen持有人。视觉测量和位置映射在≤-160℃和低的电子剂量后，冷冻水合的冷冻切片冷冻干燥在-100℃下进行约30分钟。干冰冻切片的细胞​​器级别的影像录制，也以低剂量，通过慢扫描CCD相机和选作分析的兴趣亚细胞区域的手段。由一个固定的，集中的，高强度的电子探针从感兴趣区域发射的X射线是由能量色散X射线（EDX）光谱仪，由相关联的电子处理，并提出了作为X-射线谱收集，也就是一个情节X射线​​强度与能量。其他软件方便：1）识别元素成分通过他们的“特色”峰能量和指纹，以及2）提取的峰面积/背景定量分析。本文总结了两个例子来说明典型EPMA的应用程序，其中一个线粒体钙分析提供了重要的见解兴奋性毒性损伤和其他的，揭示缺血阻力的基础机制。

脑细胞通常保持兴奋性毒性损伤的发生局部缺血条件下的病理性神经递质释放的结果。电子探针是如何发现神经元线粒体螯合钙的大量能力的基础损伤的机制是至关重要的。 图3中的电子显微照片示出了线粒体中培养的海马神经元的冻干冷冻切片30分钟后，在暴露于兴奋性刺激（100μMNMDA）快速冷冻的外观。大多数线粒体似乎是结构性损坏，因为它们是高度肿胀，含有少量，暗夹杂物（...

Watch this Scientific Journal Video about Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis at JoVE.com

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

总钙在神经元的电子探针X射线显微分析测量

本文介绍cryoanalytical电子显微镜对总钙含量和分布在生理上定义的生物标本亚细胞分辨率的定量测量中的应用。

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...