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析中的TEM。这些技术进行了简要这里描述的，但必须在其他实验室中重现这些程序的细节超出了本文的范围。有兴趣的读者可以参考最近的优秀文章9,14。 注意：本节介绍了使用的理趣efied乙烷，它是高度易燃的和潜在的爆炸性，适当应采取预防措施。运营商还必须熟悉液氮（LN 2）安全防范措施，包括防护实验室外套，眼镜，手套cryoresistant。注意：这里并贯穿始终，所有的工具（镊子等 ）用于处理冷冻样品必须在预冷2号法律公告前使用，以避免意外解冻。 盖玻片上培养的细胞<ol><li>通过在液氮冷却的中央以及冷凝气态乙烷在-160℃下准备暴跌冷冻设备。填至刻度并覆盖好与枢转铝盖在不使用时。 </li><li>用滤纸楔子，在适当的实验条件下薄水薄膜涂抹塑料盖玻片培养细胞。从边缘涂抹，以避免接触组织;理想的残留膜，通过试验和错误决定，是尽可能薄，但不能因此次在作为蒸发并允许组织表面的干燥。 </li><li>通过用夹紧钳的边缘保持盖玻片，迅速浸入液态乙烷中，可以手动或使用重力驱动的柱塞。 </li><li>将冷冻的盖玻片在附近的保丽龙碗2号法律公告，大到足以操纵盖玻片所需数量为长期储存容器。铝制旋盖罐不低于2号法律公告抢占建议。 </li></ol>培养的脑片急速冷冻<ol start="5"><li>在解剖显微镜下，查看和东方的海马脑片，不受干扰，仍然附着在文化隔膜插入。放置基准标记上的切片用黑色Sharpie笔型笔和照片的边缘附近的膜。 </li><li>仍然在显微镜下，切出约。 5×5毫米的正方形膜与围绕在广场各个切片。避免接触片或弯曲卷材Ë。 </li><li>装载的膜支撑片，通过一个¼直径琼脂垫缓冲，在自定义设计的铝制圆盘制成，以适应cryoultramicrotome（如下所述）。迅速通过气动用定制的“大满贯冻结”的设备来推动它针对液氮冷却的蓝宝石块冷冻切片。 </li><li>移动到存储所描述的培养细胞。 </li></ol> 2。 Cryosectioning 警告：成功地生产的超薄切片，干切带，而直接的和逻辑，需要培训，耐心和相当的实践。 <ol><li>烘烤配备了cryoattachment，冷却至-135℃的cryoultramicrotome </li><li>切冷冻盖玻片到约。使用锋利，预冷手术刀3×3平方毫米。嵌入件与培养朝上的粘性液体“cryoglue”（1:6混合物的乙醇和2 -丙醇11），在标准的3mm直径的铝销设计，以适应切片标本夹头。避免泄漏cryoglue到培养表面。通过降低cryobox温度≤-160℃，凝固cryoglue <ul class="lalpha"><li>冷冻的大脑切片，安全地通过螺钉领的方式直接连接光盘定制的标本夹头。 </li></ul></li><li>修剪冷冻样品选定的地区- 例如细胞丰富的盖玻片片切片或确定区域的区域-一个大约。使用金​​刚石修整工具250×250微米的块面和〜100μm的深度。 </li><li>干式切削丝带薄片在约使用35°菱形cryoknife -160℃，基本上与referencs 4,9描述。切削速度和刀具后角是凭经验确定，一个好的出发点是0.4-0.6毫米/秒，9°。公称厚度的水合部分， 即提前试片，我通常S 80纳米，虽然部分实际上是1.5-2.0倍厚，主要是由于压缩。对于满意的切片，防静电设备是必不可少的。放置的装置0.5-1厘米的刀口电离​​尖端和调整输出功率，直到部分满意丝带生产。 </li><li>用胶水（环氧树脂）睫毛木涂药棒准备睫毛探头。使用这样的探头，拿起并从刀背部分转移到辉光放电，碳包覆投过100目折叠铜网上聚乙烯醇缩醛支持电影和休息，在后面的工作架的半折叠铟箔的信封刀。倍，在电网和包络线的上半部分中，按与冷压工具。 </li><li>转移网格包裹在铝方便的格箱和存储可以在步骤1.4中所述。 </li></ol> 3。试样Cryotransfer到电子显微镜核心仪器电子探针在这个实验室是在120千伏操作，并配备用于低温显微镜，即，被设计用干净的真空中，试样面积anticontaminator，一个2K的x 2K高灵敏度数字相机和cryotransfer试样夹持器的分析电子显微镜。事先检查显微镜定位及经营状况的低收入和高倍率模式，并确认满意柱上真空，理想≤10-7乇。必要调补。 <ol><li>确认cryoholder的杜瓦组件的真空绝热是令人满意的。泵是必要的。 </li><li>冷却cryoholder到最低温度，至少-160℃，同时在显微镜载物台在高真空下，拆下固定器连接到它的控制箱的电缆。取出支架，以尽量减少LN 2蔓延到运营商和显微镜表面前测角45°顺时针倾斜。 </li><li>通过冷却的积分INSULATE准备台式cryoworkstationD罩杯为≤160°C。 </li><li>转移（quickly!）从显微镜冷却cryoholder到低温工作站。收回持有人的霜冻盾。 </li><li> 液氮储存和地方上的cryoworkstation的工作表网格夹层下检索。保持环为持有人的样本以及还放在桌子上。 </li><li>打开信封铟和移动封闭折叠网格的cryoholder的标本很好。确保电网与使用提供并关闭霜盾扳手工具扣环。 </li><li>从cryoworkstation快速去除cryoholder并插入显微镜的气闸并通过泵送次序尽快。在插入时，应该有柱真空的干扰降到最低。 </li><li>返回测角为0°倾斜。重新连接和恢复活力的控制盒，并确认样品温度≤150℃。 </li><li>笔芯的杜瓦试样夹具，并允许真空和温度才能完全恢复。 </li></ol> 4。第可视化调查<ol><li>收回持有人的霜盾揭露标本，把电子束上。 </li><li>视觉评定试样在低倍镜下，通常250X和低照明。作为如图1中所示，区段应该是薄且光滑，未折叠或重叠，平整以及连接到支撑片，一般不是由尘棒遮蔽。 </li><li>可选的拍摄选择的章节。 （注：最小化束曝光，因为冷冻水合部分是很容易受束引起的损伤。）使用自动化数字测角台存储选定部分的坐标。 </li></ol> 5。冷冻干燥的节<ol><li>冷冻干燥的部分通过增加保持器温度升高到约-100℃，〜30分钟。 </li><li> Recool持有至-160°C或以下。 </li><l我&gt;影像之前，请关闭cryoholder控制单元和物理断开控制器电缆，以避免图像漂移由于热循环和/或振动传感器。 </li></ol> 6。细胞和细胞器的成像冷冻干燥的部分结构的图像在约得到。 -160°C的低剂量，使用一个2K x 2K慢扫描CCD相机通过适当的软件控制的数字化记录的零损耗的图像。 <ol><li>激活的EM HI-MAG模式。 </li><li>在〜2000倍，并选择图像（如TIFF格式，参见图2）选择的细胞，并在高品质的部分，其位置被预先记录和存储的兴趣的亚细胞区域。 （注：将干燥后的部分，现在大大减少脆弱，不易受到电子束的损坏。 </li><li>为了选择感兴趣区域（投资回报）​​的X射线分析评估图像。此步骤可任选地脱线进行的，在这种情况下的EM可以打开关闭并温热至室温试样。 </li></ol> 7。收购的X射线谱 X射线谱可以使用任何可记录的几个商业或定制设计的X射线分析系统的最低限度地选自由能量色散型X射线（EDX）检测器，相关联的脉冲处理器的电子设备和兼容的采集和显示软件。 （本实验中所使用的系统表1所述。） <ol><li>通过将EDX检测器进柱（如果有必要），抽出物镜孔径，并插入和定心任何杂散辐射孔配置对EM的透视采集。调整cryoholder到在其中避免了试样的霜污染的最低温度，但至少低于-100℃。 </li><li>倾斜支架20°朝向探测器。 </li><li>在宽视场成像条件，并假设人是故意要分析一个individua的矩阵升线粒体，选择一个线粒体进行分析，将其移动到该领域的中心和重点。 </li><li>去点模式（在某些显微镜使用第二聚光焦点只是收敛光束），并增加了电子束电流到约 3 NA（作为衡量一个法拉第杯或类似）到100 nm的光点。 </li><li>发射光谱采集软件，并开始100秒的收购，它可以被看作活时间的显示监视器上。保存记录的谱（ 图2）。行业标准EMSA格式是首选。 </li><li>关闭电磁和多光谱文件（县）转移到（离线）分析工作站。 </li></ol> 8。 X射线光谱分析定性分析的EDX谱（ 图2，小图）在本质上是X射线强度与能量的xy图。光谱包含了有关T的元素组成的定性和定量信息他分析体积，在该峰的“特征”能识别以使峰而产生，而强度反映了该元素的量的元素。的特征峰骑在一个缓慢变化的背景下，“连续”的顶部。 （ 图2中的说明进一步讨论的EDX光谱的显着的细节）。峰值歧管的整个周期表中的能量是通过元素的公知的电子结构，因而所有的EDX软件链接到一个可以自动识别的元件数据库中定义分析物。在生理情况下，普遍关心的是适合于EDX分析的元素包括Na（Kα在1.04千电子伏），P（2.01千电子伏），K（3.31千电子伏），和Ca（3.69千电子伏）。 <ol><li>采取可用的软件库和峰匹配例程的优势，确定在光谱主要元素。在生物样品期望找到用于钠，镁，磷，硫，氯，K和Ca的峰。 （注意：最后两个元素重叠！） </li></ol>定量分析 EDX光谱的定量分析由提取的识别峰的积分面积，该值转换为浓度。用于生物分析中，建立的方式是霍尔峰/连续法4,7,10，其中利用这样的事实，该连续体（上面和图2中所定义）的强度正比于所分析的量的干质量的优势。因此，峰面积/连续面积的比例，相对于相同的比例在已知的组合物的标准光谱指定目标细胞区室中的浓度。注意，此方法提供的浓度以每重量摩尔，通常表示为毫摩尔/千克干重为单位。这个单元是不寻常的，用于解释可能需要额外的转换，例如毫摩尔/ L（湿重）或毫摩尔/毫克蛋白，如所述的埃尔sewhere 4,10。 <ol start="2"><li>提取Z = 10〜20之间的元素生物（0.5-4.0千电子伏）的峰面积（和误差估计）， 即钠，镁，磷，硫，氯，钾，钙和使用的拟合程序内置到分析一身的软件（ 见表1，特别是脚注4）。本实验采用单或多最小二乘拟合。注意，该嵌合需要小心注意解决（参见图2）K和Ca的峰之间的重叠。 </li><li>整合,1.45-1 .61千电子伏之间的连续性。替代性无干扰的区域都可以使用。 </li><li>通过比较标准计算峰/连续比率，然后浓度。整个分析中传播错误。 </li><li>使用标准的统计软件和公式来估算的平均值，反映的生物变异。 </li></ol>

<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>

作者报告没有利益冲突。

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

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。

总钙在神经元的电子探针X射线显微分析测量

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

measurement-total-calcium-neurons-electron-probe-x-ray

Research

JoVE Journal

Neuroscience

11.9K 次观看.  National Institutes of Health. 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。 

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

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

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

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

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

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

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

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

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

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

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

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 regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

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

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

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

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

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 abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

小鼠大脑中耳蜗的定位

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

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.