DJH和PAD是由澳大利亚研究理事会联动项目（LP120200081）与安捷伦科技公司和ESI有限公司BK的贡献支持由鲁尔大学研究学院PLUS，由德国卓越计划[DFG GSC 98/3]资金支持。 DJH部分是由Ramaciotti基金会的支持。 KK由西格里德Juselius基金会的支持。

本文所述程序已批准霍华德弗洛里动物伦理委员会，并坚持以动物保健的国家健康与医学研究委员会的标准。 1.分析试样的制备注:该步骤取决于样品基质进行分析而变化。 <ol><li>样品制备和切片 注:使用固定的4％多聚甲醛（PFA）和在30％的蔗糖冷冻保护在0.1M PBS中，将导致不同的来自组织的金属浸出的量。详情请见野兔等[19]。确保所有样品都发生了相同的固定和冷冻保护措施。 <ol><li> Transcardially灌注用冰冷的0.1M PBS，pH值7.4安乐死的动物（见Dodt 等 20详细方法部分），并取出大脑。 </li><li>放置脑在4％PFA O / N来修复组织。 </li> &lt;利&gt; Cryoprotect大脑通过将其放置在30％的蔗糖在0.1M PBS中24小时，然后再改变到新的30％的蔗糖另外24小时。 19 </li><li>冻结在低温恒温器的脑在-20℃放置至少1小时。 </li><li>安装在使用合适的安装介质卡盘的大脑。 </li><li>部分上使用不含金属的一次性刀片低温恒温器脑（ 例如，聚四氟乙烯[PTFE]涂层，刀具）和安装在标准显微镜载玻片。此部分的最佳厚度应为大约30微米。 </li></ol></li><li>如果使用石蜡包埋的样品，部分以所需的厚度，浮色带上温水浴和安装在标准显微镜载玻片。 注:长期固定和生物样本的石蜡包埋的确切影响还不清楚。正如在1.1中所述，确保所有的样品都发生了相同的样品制备程序，如果对比分析的目的。 <ol><li>脱蜡PA拉芬包埋标本在二甲苯中3的变化，1变更各浸渍滑动:100％的乙醇，95％乙醇，70％乙醇，并在ISO 3696或等效纯化水（以下称最小3的变化，被称为"水&#39;;看到野兔等 21进行了详细的方法）。 </li></ol></li><li>允许样品风干用于在无尘的环境中约1小时，如与在机架垂直放置的试样的滑动框和左半开盖子。 </li></ol> 2.基质匹配标准的研制注:以下是先前公布的9汇总协议。请咨询原纸上，以准备基质匹配标准组织的详细步骤。 <ol><li>获得商业头脑羊肉（或类似），并在水冲洗，清除所有血液和结缔组织。 </li><li>使用手术刀，解剖仔细大约50克皮质Tissu酒店的e和部分均匀使用手持式组织匀浆与低功率的聚碳酸酯一次性探针。划分成5个克一份，以根据不同的校准范围和期望的校准点数目。 </li><li>制备金属的溶液通过溶解每种分析物的可溶性盐（ 例如， 硫酸亚铁·H 2 O）在1％硝酸，以产生0.1，1和100mg金属毫升-1股穗每个标准。 </li><li>储液的一个预先计算的体积添加到5克等分组织（10毫克如 5微升毫升-1 10微克克-1湿纸巾的一个近似的最终浓度），以实现一个范围在加标金属水平的每个标准。 <ol><li>根据每个标准的所需最终浓度，可以使用各金属原液的组合，以确保解决方案的最小量被添加到标准。水最后秒杀添加到每个标准，以确保球菌补充液体量价出现在每个标准。 </li><li>均质低功耗的飙升标准约30秒。如果不立即使用，在-20℃保持冷冻在用Parafilm密封封端的聚丙烯管中。 </li></ol></li><li>确定准确的浓度，它可以使用以下步骤每个标准的同质性: <ol><li>微波消解<ol><li>放置6精确称量（约50毫克）的标准的等分试样在洗涤聚四氟乙烯消化容器中，并加入4毫升浓（65％）的硝酸和1毫升30％的过氧化氢。密封，并在500瓦消化30分钟。 </li><li>冷却该消化容器中，打开在通风橱并定量之后将消化溶液转移至用10mL水的等分试样的酸洗50毫升管中。使约50毫升，并准确称量最终溶液的质量。 </li><li>每个标准2.5.1.2 - 重复步骤2.5.1.1。 </li></ol></li><li>如果微波消解设备不可使用以下步骤: <ol><li>将6精确地测量（介于25 - 200毫克）的标准等分入酸洗/无金属聚丙烯管和冷冻干燥O / N。 </li><li>添加浓硝酸和热，未封端的40微升，加热块上至70℃5分钟，然后添加30％过氧化氢10μL的。热再5分钟，然后准确地使用的1％硝酸950微升1毫升总体积。 注:建议使用选择的方法，以确保消化过程是准确的消化认证的参考材料。 </li></ol></li></ol></li><li>确定在使用的标准协议通过溶液雾化ICP-MS的每个消化溶液中的金属浓度。通过确定每个等分试样之间的相对标准偏差（％RSD）评估每个标准的均匀性。确保％相对标准偏差都落在15％以内。使用EA的质量测量CH等分，计算出每个匀浆组织标准的精密金属浓度。 </li><li>返回到均质化的组织标准，包5×5mm的一次性塑料组织学模具和冻结的异戊烷在液氮中冷却。 在相同的厚度除去样品中从模具和部分上低温恒温器的冷冻标准组织块。 注:建议在不同厚度为未来的实验准备一些部分。风干标准可以无限期储存在气密无尘容器。 </li></ol> 3. LA-ICP-MS的研制分析<ol><li>地方标准和样品中的消融室中，以确保它们是装配到洛杉矶单元CCD照相机的景深内。如果仪器的调整是必要的，包括合适的参考材料（ 例如，NIST 612微量元素玻璃）。用手拧紧在房门上的两个螺丝密封日Ë消融室。 </li><li>在ICP-MS软件，在"维护"或类似的面板中选择打开的氩气阀和载气流量设定为1.2升分钟-1在适当的对话框。 注意:在这个协议中使用氩作为载气。几个例子使用任一氦或作为载气氦气和氩气的混合物。半滑舌鳎见海因里希22使用氦气和氩气混合物作为气溶胶的载气的技术细节。 </li><li>在洛杉矶软件，点击"清除"按钮来刷新氩气电池最少30分钟。 注:吹扫时间可以通过单击"清除时间"或类似的按钮来改变。当使用消融系统具有两卷消融细胞，周期性地移动所述平台的每个角落和斜对地横过该细胞，以确保尽可能多的残留的空气被从细胞尽可能除去。这可以通过选择"家庭阶段"或当量实现alent功能。 </li><li>打开的ICP-MS通过点击"等离子体上，并允许温热两小时，在此期间，步骤3.5 - 4.4可被进行。仪器设置制造商之间变化，尽管适当的LA-ICP-MS的操作条件，例如可以在兔等中找到。 10。 </li><li>组织标准代表消融<ol><li>选择行工具和大约长3毫米绘制整个组织表面烧蚀一行。 </li><li>设置的参数通过右击消融的线中的实验列表并改变下列如下:光束直径（由用户适当选择;一个30微米的正方形光束这里使用），扫描速度（光束直径的4倍每秒120微米-1）和能量密度（0.3 -0.5Ĵ厘米-2软组织;优化如有必要，更难矩阵）。在30微米的组织厚度的激光束不穿过充分THIC该组织kness，消除从显微镜支持任何潜在的污染物。正火碳23可被用来校正在组织的消融的量的变化。设置这些参数作为默认选择"默认"单选按钮，每个后续行。 </li><li>通过选择初始行，右击并选择"重复扫描&#39;复制此行六次。确保线在任一偏移的X -或由光束直径y轴。这给出了一个总的每标准七条线路，根据光束直径隔开。 </li><li>重复步骤3.5.1 - 3.5.3每个标准，确保相同长度的线。 </li></ol></li><li>要绘制消融面积超过示例，可以按照以下两种方法之一: 注:确保相同的扫描参数（光束直径，扫描速度，能量密度）用于采样线。 <ol><li>如果LA系统配备有一个宽视场中，选择线工具和从样品足够长以覆盖使用在3.5中概述的相同的激光参数在其最宽点处的样品的左上角画出一条线。重复该扫描与根据光束直径间隔的线在3.5.3概述作为根据需要许多次，以确保样品完全覆盖。 </li><li>如果宽视场不可用，确定并记录x和y坐标（通常示出的主画面为"台位置"或类似的）相应于覆盖整个样品的矩形的角落。使用这些坐标来定位消融的平行线覆盖整个样本区域中3.7.1所述。 </li><li>绘制的在后的最新标准间歇扫描线〜通过重复在3.5中描述的方法扫描样品的20小时。取决于此，可能需要多次样品的扫描持续时间。结束实验用阿迪的标准TIONAL扫描。 注意:当确定所述x和y坐标，而该细胞被清除，选择基准位置和轴的相关校准将改变先前获得用于x细节和y坐标，而差（ 即线的长度）将不受影响。 </li></ol></li></ol> 4.设置了ICP-MS的数据采集方法<ol><li>消融的标准线，由激光扫描速度划分线的长度，以确定为单行的总分析时间。重复此为样本行。 </li><li>在ICP-MS软件，创建新方法（此处显示为"批"），确保"时间分辨分析"或选择了等同。选择待检测的m / z值，然后调整积分时间为每个M / Z，以便在一个周期的总积分时间等于0.25秒。点击"保存蝙蝠CH作为"和相应的名称（ 例如，STD1）。 注:由于样本将在光束直径的4倍遍历的激光束，这确保了每个质量数据点被记录相当于光束直径12。例如，使用100微米的光斑尺寸，为400μms的扫描速度-1为0.25秒的积分时间将产生与真正的像素大小的图像。积分时间可以调整，以提高灵敏度;增加积分时间，以0.33小号扫描速度时，应放慢至光束直径的三倍。 </li><li>对于标准，进入分析时间在相应的框中的每一行扫描，外加15秒要占激光预热和清洗时间。输入样品运行列表（ 即在其中扫描的运行顺序）具有相同数目的收购（典型地按顺序编号， 即 001，002 等 ）的标准线的总数 。 </li><li>对于示例，通过保存一个替代的文件名的当前方法或分批重复用于该标准的方法中，并调整总采集时间（包括附加的15秒）和收购总数以匹配将烧蚀样品的行数。 注:由于大部分的LA-ICP-MS系统使用一个单向触发（LA触发ICP-MS），它是必不可少的ICP-MS软件的消融开始的下一行之前等待来自LA触发。在ICP-MS采集窗口将显示"等待开始"。 </li></ol> 5.运行实验<ol><li>通过加入第一种方法或分批到队列开始的ICP-MS队列，并确保该软件被从LA系统等待触发。 </li><li>在洛杉矶软件，通过点击&#39;发射&#39;使激光电源，单击"运行"，并在相应的框中激光预热时间为10秒和冲洗时间设置为20秒。 注意:此溢出会保证了ICP-MS准备开始获取新的数据时，激光烧蚀开始后面的每个行。 </li><li>通过点击&#39;开始&#39;启动激光序列。如果使用双卷细胞，确保样品杯是在适当的位置。 </li></ol> 6.定量计算标准注:对于转换ICP-MS数据转换成图像多种变化。这些措施包括使用写在开源语言的17，24，25，商业宏26和数据分析软件的国产软件工具。 7这里，使用最近开发的软件插件（在Paul 等27描述的）的基础上，一个专门的LA-ICP-MS数据分析套件28。 <ol><li>在传输所有包含运行数据（001.d，002.d 等 ）批处理文件夹复制到单独的计算机安装了分析软件。提取该批次的每一行* .csv的数据文件到一个单独的文件夹中。 注:使用脚本来自动传送* .csv的文件到一个新的文件夹，强烈推荐。见所附Python代码的更多细节。此脚本已被写入，以适应任一的Agilent 7700或8800系列ICP-MS，但是可以进行编辑，以适合从其他厂商的输出文件。 </li><li>打开软件平台，并按照标签顺序导入和分析标准如下所述产生量化图像。 </li><li>要导入从第一批的标准数据，选择"安捷伦的.csv"为"文件类型"，"整个文件夹"的"导入式"，并检查"日期格式"是用电脑格式相同。点击"导入"，选择包含.csv的文件的第一套标准的文件夹，并移动到"基线"选项卡。 </li><li>底线减法<ol><li>使用从工具栏的主应用程序下拉菜单的"自动选择"选项卡（命令2），选择"从导入信息"，然后点击"继续"。 </li><li>点击"全选"，然后选择"Baseline_1"进行整合。 </li><li>选择第一个10秒每条扫描线（对应于激光预热时间）中，输入第二个值"0"和作物的数据"（行的时间-第10号）"，然后单击"添加Integrations公司（ 如，对于一个35秒的扫描线，输入值&#39;0&#39;和&#39;25&#39;）。 </li></ol></li><li>要选择样本数据，使用"自动选择"选项卡，在6.4描述，但选择"OUTPUT_1"作为整合。从一开始，每个标准行结束作物数据排除背景信号（用于一个35秒的扫描线例如 ，输入值"13"和"4"）。 </li><li>以排除液滴造成possi信号在标准BLE孔，在"样品"选项卡，单击图形区域的左上角"渠道"，选择具有高信噪比（如 C13或P31）的通道。请记在信号的急剧下降，并选择一个CPS值足够低，是样品中的正常变异的下方，但足够高，以挑选出的信号的急剧下降。 </li><li>点击"DRS"选项卡，然后选择"当前数据缩减计划&#39;Baseline_Subtract"。选择6.6"指数频道"的频道和&#39;屏蔽低信号时，门限用&#39;的CPS值。插入一个值&lt;0.5秒&#39;秒钟，然后修剪/低后信号&#39;以考虑从激光到ICP-MS信号传输的延迟。 </li><li>要确认低信号区足够的遮蔽，单击后退到"样品"选项卡并选择处理（ 例如 ，Fe56_CPS）通道轨迹。重复和完善CPS和＆＃39，秒修剪前/低信号的价值观后，在6.7，如果必要的。 </li><li>点击"结果"选项卡，并从主应用程序的下拉菜单中选择"导出数据"。输入一个文件名来生成结果的电子表格数据文件（ 例如，STD1计算，STD2 ...）。 </li><li>开在合适的电子表格程序中的数据文件，并计算CPS值来进行定量每个单独的信道。使用从溶液雾化的ICP-MS的预定金属浓度从CPS值计算的转换系数到ppm（微克g -1）以上的每个量化的元件。 </li><li>重复步骤6.3 - 6.10对所有套在运行标准。 </li></ol> 7.构建定量图片<ol><li>写&#39;Biolite（）&#39;在命令提示符中打开该软件。 </li><li>通过点击"载入图片"，然后选择文件夹包含.csv的文件导入FO样本数据r为样本图像。 </li><li>背景校正<ol><li>点击"基准"选项卡应用背景校正的样品。在磷（P31）影像提示在屏幕上，使用矩形工具绘制背景的选择区域。选择尽可能多的区域尽可能将创建信号/等离子体漂移的图象全图，以确保从这些干扰因素足够的补偿。 </li><li>为了使背景信号更加明显，选择图形编辑工具（左上方显示的图像），并右键单击图像。选择"修改图像外观"和"在Z =首先颜色"变更为较大的负值（ 如 -100,000）。点击"完成"继续。 </li></ol></li><li>点击"标准"选项卡，弹出一个表CPS / ppm的校正因子。在步骤6.10的每个元件的输入与标准计算值。这一步骤将有助于以校正在ICP-MS的灵敏度漂移。点击"开始！ </li><li>从"数据"选项卡，其中包含每个元素和每一步的图片打开"数据浏览器"。 </li><li>要完成出口图像，请在"StdCorrImages"文件夹中所需的图像，用鼠标右键单击图像名称（* _ppm），然后单击"NewImage"。右键点击图片打开"修改图像外观"来选择所需的颜色表和色阶。通过右键点击图片，选择"添加注释"色标添加到图像。从左上角的下拉菜单中选择"ColorScale"，并使用选项卡来修改所需的色阶。 </li><li>要导出图像，确保有问题的图像被选择并导航到"文件"选项卡，然后单击"保存图形..."。选择所需的格式并保存图像。另外，所选择的图像可以使用复制和粘贴工具进行传输。 </li><li>重复步骤对所有感兴趣的图像7.6和7.7。 </li><li> Quantif英离散区域<ol><li>要激活ROI工具，导航到"分析"选项卡中，"包"，选择"图像处理"。 </li><li>选择所需的图像，然后导航到"图片"选项卡，点击"投资回报率......"。点击"开始抽奖的投资回报率"，并使用绘图工具来选择样本中的感兴趣区域。要完成图形中，单击"完成的投资回报率"。 </li><li>为了获得所选择的投资回报率的统计数据，导航到"图像"选项卡并单击"统计..."。 </li><li>复制并粘贴结果到一个单独的电子表格。重复投资回报率选择（7.9.2）的所有地区和感兴趣的内容。 </li></ol></li></ol>

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RWH and COC are paid employees of ESI Ltd. FF and TPH are paid employees of Agilent Technologies Australia.

在神经组织成像的金属是如何这个协议可提供金属的任何生物基质中的分布和量有用的信息的一个例子。虽然编制标准参考材料可以是艰巨的，它是一个可以一次执行和存档以供日后使用一个实验。 LA-ICP-MS具有比其他方法，例如基于同步加速器X射线荧光显微镜，大多在可访问性和灵敏度方面的某些优点。然而，也有应制备使用LA-ICP-MS实验时，应考虑某些缺点，因此它通常是化学成像，其包括替代金属分析技术，以及比较组织化学5的有用补充技术。 对准用鼠脑的已知解剖特征可以提供关于可能的功能relati有用信息金属含量和空间分布之间onship。此前，我们已经使用了艾伦脑图谱在线资源，29这是在C57BL / 6小鼠大脑既解剖和基因表达数据的开放获取资源库研究金属依赖性酶的表达14和神经解剖学27两者的空间相关性， 30。其他资源，如鼠类脑工作台31也可与登记和金属图像的比对，以帮助以协助小常解剖区域正确识别的金属分布。 这种技术的应用是在微尺度整个正常的生活事件评估如何金属含量和分布的变化非常有用（如老化），并在疾病状态;以及研究设计既含金属化合物和药物的效果为目标我TAL新陈代谢。 LA-ICP-MS的作为用于在空间上评估金属分布的成像技术当前的主要限制是可以通过和灵敏度。有分析速度和空间分辨率5,12之间的折衷，与需要较长的分析时间分辨率较高的图像。该技术是适合于在较高浓度的生物分子，虽然元素如锰，钴和硒由于它们的低丰度，在它们的检测通过常规的ICP-MS的正常组织和/或限制的约束。在ICP-MS技术的新进展，如推出三重四极杆质谱分析仪，允许在较高的感光度33难分离的物质，如硒32的有针对性的检测。作为一家技术驱动程序，先进的激光和质谱仪的设计会看到这样的成像技术的不断发展，越来越多分析和灵敏度34的速度。

金属的独特的氧化还原化学促进一系列神经功能，包括信号转导，能源生产和神经递质的合成。在一些重大的神经变性疾病的，这些金属的dyshomeostasis已经在疾病的发病机制都牵连并鉴定为治疗干预1潜在的新靶标。为了更好地了解金属如何参与如（分别为AD和PD，）阿尔茨海默氏症和帕金森氏症的条件下，就必须能够测量金属分布和水平的疾病进程产生不利影响的区域内如何变化。这些变化往往在指示可密切联系到引发细胞死亡，如我们在PD 2铁和多巴胺的神经毒性最近提出的机制过程中的生化反应的微妙变化中。 传统上，荟萃定义解剖区域内的L层已经通过仔细切除，消化和分析使用的分析技术3实现。然而，这样的方法丢失空间信息，当正在研究的疾病状态涉及小的，明确定义的区域或特定的细胞类型可以是至关重要的。一些分析方法可用于可视化在生物系统的金属，从完整样品组织切片，并在二维和三维，使用发射光谱，荧光探针和质谱4。每种技术具有关于灵敏度，化学物质的选择性，并且能够实现空间分辨率的优点和缺点。对于现有技术的范围进行了全面概述，看到野兔等人的审查。 5。 质谱（MS）为基础的方法是最灵敏的这些技术，能够在家乡浓度6测量生物最相关的金属。激光烧蚀 - 电感耦合等离子体 - 质谱（LA-ICP-MS）的成像采用聚焦的紫外线激光束大小不等，从1至&gt; 100微米的直径（或宽度，当使用一个四边形的光束形状），在其下样品传递7。可以通过标准参考材料代表消融，其可以使用各种不同的方法如图8所示 ，每个都有不同程度的技术难度和分析实用性来生产来实现的定量信息。最常见的方法是使用矩阵的匹配，其中，用一主要化学组成媲美的样品的标准通过与靶分析物扣球制备准确地由独立的分析装置9评估均一性的和绝对的金属浓度， </suP&gt; 10。的准备的标准消融然后可用于外部校准目的，使每个像素将被提取从所得的样本图像浓度的数据。 图像分辨率由束尺寸和在该样品的扫描速度两确定。标准的四极设计的ICP-MS（占全世界所有已安装的ICP-MS系统11的超过90％）是一个顺序质量分析器，在通过所有选定质荷比的质量检测器的周期（M / Z ），而不是同时收集数据。因此，采集时间为群众的每个循环必须等同于遍历的激光束中的一个的宽度，以确保一个象素代表所期望的分辨率的取得12对样本采取的时间。激光束尺寸选择为具有两个灵敏度和总的分析时间显著效果的关键参数。激光烧蚀physi卡利移除由氩载气扫至ICP-MS的材料，可以由该质量分析器被物理检测的物质的量如下平方反比定律。通过四个因素为25μm导致减少烧蚀材料 - 例如，减少为50的激光束的直径。此外，作为扫描方法，更小的光束直径增加以消融所选择的区域所需的总时间。因此，实验设计是至关重要的，以平衡与灵敏度的需求和时间限制的必要的空间分辨率。 成像通过LA-ICP-MS已经被应用到一个范围的样品，矩阵和疾病状态，包括神经系统紊乱13，14，外伤性脑损伤15，在胎盘的抗癌药物16含金属的，毒物曝光分布的动物模型17和金属颇ibution在牙齿生命早期的膳食变迁的生物标志物。 18在这个协议中，我们描述的一般方法为在30微米的分辨率在WT小鼠大脑成像铁，铜和锌，尽管它可以很容易地适应各种样品类型和实验结果的基础上，在需要的分析师。

<table><tbody><tr><td>Soda glass microscope slides</td><td>n/a</td><td>n/a</td><td>Typical slides are suitable for all experiments</td></tr><tr><td>PTFE-coated microtome blades</td><td>C.L. Stuckey</td><td>DT315R50</td><td>Blade size depends on cryostat blade holder. Check before ordering.</td></tr><tr><td>Parafomaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>16005</td><td>Any supplier suitable</td></tr><tr><td>Sucrose</td><td>n/a</td><td>n/a</td><td>Commercial grade white sugar is suitable</td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>P5368</td><td>Premixed sachets listed, can be prepared according to normal laboratory protocols</td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>247624</td><td>Any supplier suitable</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Any supplier suitable</td></tr><tr><td>Lamb brain</td><td>n/a</td><td>n/a</td><td>Available from most local butchers</td></tr><tr><td>Metal salts</td><td>n/a</td><td>n/a</td><td>Use water soluble metal salts containing desired analytes</td></tr><tr><td>Omni TH Tissue Homogeniser</td><td>Omni Inc</td><td>THP115</td><td>Alternative homogenizers are suitable</td></tr><tr><td>Polycarbonate homgenizer probes</td><td>Omni Inc</td><td>TH115-PCRH</td><td></td></tr><tr><td>Microwave digestion unit</td><td>n/a</td><td>n/a</td><td>Optional. See Section 2</td></tr><tr><td>1.5 mL microfuge tubes</td><td>TechnoPlas</td><td>P4010</td><td>Metal-free polypropylene tubes. Acid washed tubes are also suitable</td></tr><tr><td>65% nitric acid</td><td>Merk Millipore</td><td>100441</td><td>Trace analysis grade</td></tr><tr><td>30% hydrogen peroxide</td><td>Sigma-Aldrich</td><td>95321</td><td>Trace analysis grade</td></tr><tr><td>10 x 10 mm disposable cryomolds</td><td>Ted Pella</td><td>27181</td><td></td></tr><tr><td>Isopentane</td><td>Sigma-Aldrich</td><td>76871</td><td></td></tr><tr><td>Liquid nitrogen</td><td>n/a</td><td>n/a</td><td>Use local supplier</td></tr><tr><td>NWR213 Laser Ablation system</td><td>ESI Ltd</td><td>n/a</td><td>Used in these experiments. Other manufacturers suitable, may require modifications to protocol</td></tr><tr><td>Agilent 8800 Series ICP-MS</td><td>Agilent Technologies</td><td>n/a</td><td>Used in these experiments. Other manufacturers suitable, may require modifications to protocol</td></tr><tr><td>Iolite</td><td>Iolite Software</td><td>n/a</td><td>Available from http://iolite-software.com/. Other methods are available, see protocol</td></tr><tr><td>Excel</td><td>Microsoft</td><td>n/a</td><td></td></tr><tr><td>IGOR Pro</td><td>Wave Metrics</td><td>n/a</td><td>Avalable from https://www.wavemetrics.com/products/igorpro/igorpro.htm</td></tr></tbody></table>

imaging metals in brain tissue by laser ablation - inductively coupled plasma - mass spectrometry (la-icp-ms)

金属在整个生物体中无所不在，用自己的特定解剖区域内的两种化学活性和数量决定的生物学作用。内的脑，金属有一个高度条块分配，这取决于它们对中枢神经系统中发挥主要作用。成像金属的空间分布提供了独特的见解入脑的生化结构，允许神经解剖学区域及其对于金属依赖性过程的已知功能之间的直接相关性。此外，若干与年龄相关的神经障碍功能部件打乱金属动态平衡，这通常限于那些否则难以分析大脑的小区域。在这里，我们描述了一个全面的方法在小鼠大脑成像定量金属，使用激光烧蚀 - 电感耦合等离子体 - 质谱（LA-ICP-MS）和特别设计的图像处理软件。着眼于铁，铜和锌，这三种大脑内的最丰富和疾病相关的金属，我们描述了样品制备，分析，定量测量和图像处理的基本步骤，以低微米内产生金属分布图分辨率范围。这种技术，适用于任何切割组织切片，能够表现出一个器官或系统内的金属的高度可变分布的，并且可以用于识别在金属体内平衡和优良的解剖结构内的变化的绝对水平。

为了证明这一点的LA-ICP-MS成像方法的能力，使用WT C57BL / 6小鼠大脑的单个部分，在胼胝体等分并切片在冠状平面内的简单的实验，呈现。作为在所分析的部分（ 图2）提供金属分布的代表图像在第6和7描述的，以及使用Biolite（ 图1）的数据进行分析的工作流，也被描述。 如可以看到的那样，在小鼠脑中金属分布根据解剖区域是可变的。这可以归因于可变角色金属，以及它们所结合的更具体的蛋白质，起到各脑区域27。例如，铁倾向于具有更高的浓度在脑和沿齿状回，而锌是最丰富的科尔蒂CAL领域。碳，这是一种常用内标8，是均匀分布的。元素映射在与现有的解剖和功能参考地图集29，其中对金属的共定位信息可以特定金属结合蛋白质的表达可以在大脑内提供的洞察金属的功能一起使用时（ 图2）可以是特别有用区域，或与所识别的疾病相关的生物分子的变化在金属水平的线。使用所描述的方法，这是对更广泛的分析物的最佳化，确实排除对低丰度的元素如锰灵敏度和方法可以适用于由其他测量质量为代价增加驻留时间主要集中在这个分析物。 在使用通过LA-ICP-MS成像的主要优点是观察在金属精矿相对差异通货膨胀和实验组之间分配。我们以前曾使用这种技术来证明下面的神经毒素损伤模拟PD 2,10增加铁，而在人类阿尔茨海默氏病组织21皮质铁水平的变化。如这里描述的这样的协议可以很容易地适于以最小的修正列出的方法的任何其他组织类型。 <img alt="图1" src="/files/ftp_upload/55042/55042fig1.jpg" /> 图1: 工作流的图像处理。工作流第6和7中，描绘了在小鼠大脑金属分布的定量图像从ICP-MS的原始时间分辨数据的转换是互补的。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55042/55042fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图2" src="/files/ftp_upload/55042/55042fig2.jpg" /> 图2:Biometals 小鼠大脑内的典型元素分布。与LA-ICP-MS分析单个小鼠大脑半球的30微米厚的冠状切面的代表元素映射。对于碳13（C13），镁-24（MG24）和磷-31（P31）影像显示的黄金色标（每秒计数; CPS）（上排）。锰-55（Mn55），铜- 63（Cu63），铁- 56（Fe56）和锌- 66（Zn66）定量图像（底行）与对应BlueHot色阶（微克g -1）以上显示。总分析时间为大脑部分约为5小时。比例尺= 2毫米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55042/55042fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <strong&gt;补充代码文件中描述的6.1 1.开源Python代码，它可以使用适当的Python编辑器根据数据输出的ICP-MS制造商的方法进行修改。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55042/CopyFilesFromSubDirs_3.txt" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS at JoVE.com

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

通过激光烧蚀定量映射在组织金属 - 电感耦合等离子体 - 质谱（LA-ICP-MS）是一个灵敏的分析技术，可以提供新的见解金属如何参与正常功能和疾病过程。在这里，我们描述了在小鼠神经组织的超薄切片定量成像金属的协议。

在脑组织成像金属激光剥蚀 - 电感耦合等离子体 - 质谱法（LA-ICP-MS）

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

imaging-metals-brain-tissue-laser-ablation-inductively-coupled-plasma

Universidad San Sebastian

Research

JoVE Journal

Medicine

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

21.5K 次观看.  University of Technology Sydney. 通过激光烧蚀定量映射在组织金属 - 电感耦合等离子体 - 质谱（LA-ICP-MS）是一个灵敏的分析技术，可以提供新的见解金属如何参与正常功能和疾病过程。在这里，我们描述了在小鼠神经组织的超薄切片定量成像金属的协议。 

视频: Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

科研

工程学

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

生物化学

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

生物工程

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves several steps, include drawing samples, cooling, cutting, transport to the laboratory, and analysis. The whole analysis process requires more than 30 minutes, which hinders on-line process control. Laser-induced breakdown spectroscopy is an excellent on-line analysis method that can satisfy the requirements of vacuum induction melting because it is fast and noncontact and does not require sample preparation. The experimental facility uses a lamp-pumped Q-switched laser to ablate melted liquid steel with an output energy of 80 mJ, a frequency of 5 Hz, a FWHM pulse width of 20 ns, and a working wavelength of 1,064 nm. A multi-channel linear charge coupled device (CCD) spectrometer is used to measure the emission spectrum in real time, with a spectral range from 190 to 600 nm and a resolution of 0.06 nm at a wavelength of 200 nm. The protocol includes several steps: standard alloy sample preparation and an ingredient test, smelting of standard samples and determination of the laser breakdown spectrum, and construction of the elements concentration quantitative analysis curve of each element. To realize the concentration analysis of unknown samples, the spectrum of a sample also needs to be measured and disposed with the same process. The composition of all main elements in the melted alloy can be quantitatively analyzed with an internal standard method. The calibration curve shows that the limit of detection of most metal elements ranges from 20-250 ppm. The concentration of elements, such as Ti, Mo, Nb, V, and Cu, can be lower than 100 ppm, and the concentrations of Cr, Al, Co, Fe, Mn, C, and Si range from 100-200 ppm. The R2 of some calibration curves can exceed 0.94.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

激光击穿光谱对真空感应熔融的定量分析

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

癌症研究

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

生物学

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

神经科学

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

<meta charset="utf8"></head><body> 脑类器官中代谢物的高级 MALDI-MSI 映射

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry 

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

<meta charset="utf8"></head><body>使用质谱法对人脑类器官进行分子成像

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.