这项工作得到了国家科学中心（NCN）在2018/31/D/ST5/00399和OPUS10 2015/19/B/ST7/02163项目SONATA14的部分支持。

1.缺陷选择性蚀刻
<ol><li>固体蚀刻剂制备<ol><li>通过将组成碱氢氧化物和金属氧化物在蒸馏水中溶解和混合，制备氢氧化钾 （KOH） 和氢氧化钠 （NaOH） 强碱与氧化镁 （MgO） 的共晶混合物。分别在 NaOH、KOH 和 MgO 的百分比下保持 53.6/37.3/9.1 的化学计量量13.MgO的添加增加了蚀刻剂的粘度，使其保持在Ga极性表面上，并且不会流过边缘流向N极性表面13,14。所有使用的化学品都应具有实用级质量。</li>
		<li>将热板上的烧瓶中的混合物加热至200°C，并通过磁力搅拌搅拌1小时（高于KOH-NaOH共晶点的熔点）。</li>
		<li>通过降低热板的温度将混合物冷却至~100°C，以完全蒸发剩余的液体。此步骤取决于烧瓶大小和水量，因此可能需要几分钟长达 1 小时。</li>
		<li>将固体蚀刻剂（表示为 E+M）转移到干燥的瓶子中，避免暴露在湿气中。 注意:KOH 和 NaOH 可能会引起皮肤刺激和眼睛损伤。戴上手套和护目镜。该协议也可以在此处暂停。</li>
	</ol></li>
	<li>缺陷选择性蚀刻<ol><li>准备干净的氮化镓表面进行分析。在蓝宝石上外延生长的 GaN 用于以下步骤12.</li>
		<li>将GaN样品放在加热至~450°C的热板上。 将热电偶放在样品附近，以精确读取真实温度。</li>
		<li>将一块固体 E+M 蚀刻剂放在 GaN 顶部并静置 3 分钟。</li>
		<li>从热板中取出样品，放入装有热HCl的烧杯中3-5分钟，以消除剩余的E + M。</li>
		<li>从HCl中取出样品，放入装有去离子（DI）水和超声波浴的烧杯中5-10分钟。</li>
		<li>用 N2 吹干样品。 注意:盐酸可能会引起皮肤刺激和眼睛损伤。戴上手套和护目镜。避免被烫伤。该协议也可以在此处暂停。</li>
	</ol></li>
</ol>2. 扫描电子显微镜（SEM）观察
<ol><li>在样品上做标记（例如，使用金刚石裁刀用L形划痕）。</li>
	<li>使用导电粘合剂（即双面碳导电胶带或类似材料）将样品安装在专用于要使用的 SEM 模型的金属短截面上。在样品制备和转移过程中戴上手套，以避免手上的油脂污染。</li>
	<li>添加步骤 2.2 中的一块胶带，将样品表面与金属短截线连接起来，以防止电荷在样品表面积聚。或者，可以应用带有导电材料（~10 nm 厚）的溅射涂层以防止充电效应。</li>
	<li>获取至少三张高分辨率 SEM 显微照片（理想情况下，至少五张）样品的俯视图。每张图像应显示至少 25 x 25 μm 的面积。避免从具有宏观表面缺陷的表面区域拍摄图像。 图 1 给出了一个典型的结果。</li>
	<li>请注意每张图片相对于 L 形标记的确切位置。 注意:协议可以在此处暂停。</li>
</ol>3. 二次离子质谱测量
<ol><li>工具校准<ol><li>使用负极性、Cs 初级离子以 7-13 keV 冲击能量校准 SIMS 设备。对齐次梁和主梁。保持光束尽可能小（直径至少 1 μm），因为横向分辨率由光束的大小预先确定。</li>
		<li>为具有不同离子电流密度的光束准备五到七个设置。为简单起见，请保持光束的大小不变，并更改光束电流。测量光束电流和光束15 的大小。在以下步骤中，使用5 nA、10 nA、15 nA、20 nA、30 nA和50 nA的束流，光斑尺寸为1 μm。</li>
		<li>使用 50 x 50 μm 光栅大小和 35 x 35 μm 分析区域执行以下步骤。选择 256 x 256 像素以获得空间分辨率。如果未另行指定，则对每个信号使用标准积分时间（通常为 1-2 秒）。</li>
	</ol></li>
	<li>主要来源稳定性<ol><li>选择具有中等束流 （15-20 nA） 的设置。</li>
		<li>使用 30Si2- 二次离子获得空白硅晶圆的一系列图像。对于每个图像，将信号积分 5-10 分钟。</li>
		<li>将所有图像与第一张图像进行像素间比较。如果 >5% 的像素与第一张图像相差 >5%，则表明光束变得不稳定。注意光束稳定性的时间跨度。</li>
	</ol></li>
	<li>测量 注意: 以下步骤是在光束的稳定时间跨度内执行的。<ol><li>按照 Peres 等人描述的程序估计测量室7 中氧气污染的背景水平。对于每次测量，无需获得氧浓度的绝对值，因为 16个 O- 和 69个 Ga- 信号的强度比就足够了。</li>
		<li>使用在步骤 3.1.2 中准备的光束设置。对每个光束设置至少执行五次测量。使用16 O-二次离子获得深度分布，达到~200 nm深度，并通过对信号进行10-15 s的积分来测量69Ga-二次离子的强度。不要在已获得 SEM 图像的区域执行此操作。</li>
		<li>绘制 16 个 O 和 69个 Ga- 信号的强度比作为反转初级电流密度的函数（无需计算绝对值）。预期有良好的线性拟合（此处，R2 = 0.997）。估计真空背景贡献，如 图 2 所示。</li>
		<li>为以下步骤选择强光束 （30 nA）。获取将用于平场校正的图像。将 30Si2- 二次离子用于空白硅晶圆。对信号进行积分 5-10 分钟。 图 3 给出了一个典型的结果。</li>
		<li>在获取 SEM 图像的相同区域执行深度剖面测量。使用 16O- 二次离子，对每个数据点的信号进行 3-5 秒的积分。 注意:协议可以在此处暂停。</li>
	</ol></li>
</ol>4. 数据处理
<ol><li>从深度剖面重建 3D 图像。</li>
	<li>执行平场校正:使用在步骤3.3.4中获得的参考图像对每个 16O- 离子图像进行像素到像素归一化。图 4A 为原始数据， 图4B 为平场校正后的图像。</li>
	<li>从步骤3.3.2中获得的图中估计真空背景贡献。无需计算绝对值;但是，请注意可归因于真空背景贡献的总计数的特定百分比。对于这种实验，通常介于 90-95% 之间的值。</li>
	<li>减去真空背景贡献:随机消除 90-95% 的 16 个 O 计数。 图4C 给出了单个平面的典型结果。</li>
	<li>将剩余计数绘制为 3D 图像。 图 5 给出了一个典型的结果。</li>
	<li>使用任何支持图层的图像编辑器软件，集成来自所有数据点的信号，并将 2D 图像叠加在先前获得的 SEM 图像上。将 SEM 图像视为背景。包含 SIMS 结果的图层应仅包含彩色像素的实际计数（删除中间的白色区域）。向此图层添加 ~30% 的透明度。 图 6 显示了一个典型结果</li>
</ol>

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作者没有什么可透露的。

探测器的不均匀性和真空背景贡献问题很容易通过平场校正和寄生计数的减法来解决。减法程序并不完美，因为它可能会减去氧气聚集的贡献。相反，在另一个位置，它将使背景计数不受影响;因此，一些人为计数可能仍然存在，而一些实际计数会减少。然而，它足够高效和灵敏，可以提供可接受的结果。
初级光束不稳定性是最成问题的，因为初级光束参数的劣化会使二次离子图像模糊;因此，无法获得有关样品的可靠信息。协议中的第3.2节尤为重要。例如，对于对准良好的光束，第一个 30Si2- 二次离子图像反映了检测器的不均匀性，但一段时间后，图像将开始发生变化。这是由初级光束参数的劣化（即初级电流损耗、散焦、位置漂移等）引起的。因此，估计光束稳定性的时间跨度非常重要。建议在光束初始化后 2-3 小时开始实验，因为它通常更稳定。
如果实验是在光束的稳定时间跨度内进行的，但结果仍然不令人满意，建议考虑主光束的质量。对于较小的初级光束，仅通过观察二次离子图像来确认足够的质量更具挑战性。因此，建议在溅射~1μm非常平坦的材料（即空白硅晶圆）后，在陨石坑底部进行原子力显微镜粗糙度测试。如果均方根粗糙度大于 1 nm，则需要进一步优化主光束。
光束的大小限制了该方法的横向分辨率。SIMS可以对比束尺寸小的特征进行成像，但二次离子图像将继承一次离子束的形状和大小。如果两个特征之间的距离小于光束的大小，二次离子图像将使它们模糊在一起。尽管存在这些问题，该方法仍允许用户获得固态样品中杂质/掺杂剂的真实 3D 分布。此外，原子的任何空间偏析都可以与缺陷和界面的位置相关联。
对于基于 GaN 的结构（即氧装饰），作为局部非辐射复合中心的位错负责 n 型电导率。对于其他材料，掺杂剂/污染原子分布的任何不均匀性都可能对器件的性能产生重大影响。因此，该协议对于生长和处理程序的故障分析和优化特别有用。

二次离子质谱（SIMS）是一种用于污染监测的知名技术，具有出色的检测限1,2,3,4,5,6。对于轻元素（例如氢、碳、氮、氧）来说，真空背景贡献可能是有问题的，这些元素可能以残余气体的形式存在于测量室中。Peres等人之前建立了一种估计背景贡献的技术;因此，可以确定污染原子的实际浓度7.
在许多材料中，污染原子的分布并不均匀。氮化镓（GaN）的情况特别有趣，因为据预测，氧主要装饰螺杆和混合位错8,9,10,11。考虑到大多数分析方法缺乏检测低浓度污染原子的灵敏度或空间分辨率，因此必须开发一种能够对分离杂质进行 3D 定位的 SIMS 测量程序12。
虽然许多SIMS光谱仪都配备了位置敏感探测器，但深度剖面的直接三维（3D）重建不足以获得氮化镓样品中氧原子的真实分布。探测器的缺陷可能会使图像失真，并阻止研究人员获得污染原子的真实分布。然而，一个很大的问题是真空背景贡献，因为通常>90%的登记氧计数来自分析室中存在的残余气体。这里介绍的是一种识别和充分解决这些挑战的方法。
探测器的不均匀性可以在空白硅晶圆上进行测试。由于微通道板检测器中每个通道的灵敏度不同，即使是较长的积分时间也会导致观察到一些二次离子图像的不均匀性。因此，需要平场校正来获得偏析原子三维分布的高质量图像。
真空背景贡献与从真空吸附到分析区域的污染原子通量有关。考虑到该过程是动态的（即样品表面不断被主光束溅射），可以假设分析区域的每个点吸附这些氧原子的概率相同。此外，它们几乎立即被溅射，没有足够的时间进行分离。因此，统计方法是最有效的。随机消除 90%（或更多）的氧计数应揭示氧聚集的区域。
应该注意的是，主光束的稳定性对于这种类型的实验至关重要。一段时间后，光束的强度和均匀性会变差，从而降低图像质量。因此，必须估计光束稳定运行的时间跨度，并在光束变得不稳定之前进行所有实验。该协议可以很容易地用于其他材料和检测预计分布不均匀的元素。将其与湿化学蚀刻相结合特别有趣，湿化学蚀刻揭示了位错的位置和类型。因此，团聚杂质的位置可以与缺陷的位置相关联。

<table><tbody><tr><td>Heating plate with ceramic top plate</td><td>IKA - Werke GmbH</td><td>3644200</td><td>for defect selective etching; yellow MAG HP 7</td></tr><tr><td>Hydrochloric acid (HCl) solution 35-38%</td><td>Chempur</td><td>115752837</td><td>for etchant removal; pure p.a.; CAS: 7647-01-0</td></tr><tr><td>Magnesium oxide (MgO)</td><td>Chempur</td><td>116140200</td><td>for eutectic solid etchant prepration; pure p.a.; CAS: 1309-48-4</td></tr><tr><td>Potassium hydroxide (KOH)</td><td>POCH S.A.</td><td>746800113</td><td>for eutectic solid etchant prepration; pure p.a.; CAS: 1310-58-3</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>POCH S.A.</td><td>810925112</td><td>for eutectic solid etchant prepration; pure p.a.; CAS: 1310-73-2</td></tr><tr><td>Secondary ion mass spectrometer</td><td>CAMECA</td><td>IMS SC Ultra</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Hitachi</td><td>SU8230</td><td></td></tr></tbody></table>

3d depth profile reconstruction of segregated impurities using secondary ion mass spectrometry

所提出的方案结合了使用二次离子质谱（SIMS）的出色检测限（1 ppm至1 ppb）和合理的空间分辨率（~1 μm）。此外，它还描述了如何获得固态材料中分离杂质/掺杂剂的真实三维 （3D） 分布。由于与SIMS相关的测量伪影，通常很难实现直接的3D深度剖面重建。这里介绍的是一种识别和解决这一挑战的方法。讨论了三个主要问题，包括 i） 通过平场校正补偿探测器的不均匀性;ii） 估计和减去真空背景贡献（分析室中存在的残余气体中的寄生氧计数）;iii）在一次离子源的稳定时间跨度内执行所有步骤。湿化学蚀刻用于揭示材料中位错的位置和类型，然后将SIMS结果叠加在通过扫描电子显微镜（SEM）获得的图像上。因此，团聚杂质的位置可能与某些缺陷的位置有关。该方法速度快，不需要复杂的样品制备阶段;然而，它需要高质量、稳定的离子源，并且必须快速进行整个测量，以避免主光束参数恶化。

在3D图像中应观察到非常清晰的柱状结构。更多的氧气应该聚集在靠近表面的区域，因为蚀刻过程会引入更多的氧气，这些氧气可以通过样品扩散。 图 7 显示了原始数据的 3D 图像和还原过程如何显示最终结果的动画。 图4C 还给出了单个平面的典型结果。
叠加在SEM图像上的SIMS图像显示，氧气沿着最大蚀刻坑的岩心聚集。这些可归因于混合/螺钉位错15。需要注意的是，如果核心小于主光束的尺寸，则次映像将继承主光束的尺寸和形状。在次优实验中，可以看到氧计数的随机分布（图8）。 图 9 显示了光束在实验过程中变得不稳定的情况。具体来说，对于如此接近地表的区域来说，质量很高，但在实验过程中会逐渐恶化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61065/61065fig1.jpg"> 图 1:使用 E+M 蚀刻在 GaN 表面显示的蚀刻坑的 SEM 显微照片。 将蚀刻参数设置在450 °C下3分钟。插图描绘了一张放大的显微照片，揭示了位错岩心上产生的六边形凹坑。两个最大的凹坑 （>500 nm） 表示具有 Burgers 矢量螺杆分量的位错。此图经许可转载12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61065/61065fig2.jpg"> 图2:平均O-浓度与反向初级电流的关系。 真空背景贡献可以从图中估计出来。误差线表示每个数据集的标准偏差（五个测量值）。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61065/61065fig3.jpg"> 图 3:空白硅晶圆的典型 30Si2- 二次离子图像。 强度差异是由探测器的不均匀性引起的。<a href="https://www.jove.com/files/ftp_upload/61065/61065fig3large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61065/61065fig4v2.jpg"> 图 4:在 3D 模式下测量的氧计数分布的典型平面图。 显示的图像 （A） 来自原始数据，（B） 平场校正后，以及 （C） 减去真空背景贡献后的图像。此图经许可改编12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig4large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61065/61065fig5.jpg"> 图 5:5 μ m x 5 μ m x 1 μm 长方体中氧气计数的 3D 视图。 为了获得更好的可见性，z 刻度被拉长。有关动画，请参阅补充图 1。此图经许可改编12.<a href="https://www.jove.com/files/ftp_upload/61065/61065fig5large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61065/61065fig6.jpg"> 图6: 投射在SEM显微照片上的氧二次离子（蓝色像素）的横向分布。 尽管存在与SIMS相关的伪影（由主光束的大小决定的横向分辨率），但观察到最大凹坑的位置与氧气之间存在明显的相关性。此图经许可改编12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig6large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61065/61065fig7.jpg"> 图 7:显示如何执行还原过程的动画。 在程序开始时，所有计数都存在，然后对于每一层，随机消除 90% 的计数。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig7.mp4" target="_blank">请点击此处下载此动画。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61065/61065fig8.jpg"> 图8:次优实验中氧计数的随机分布。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig8large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61065/61065fig9.jpg"> 图 9:使用不稳定光束进行的实验。 质量随着溅射深度的增加而下降。<a href="https://www.jove.com/files/ftp_upload/61065/61065fig9large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图 1. <a href="https://www.jove.com/files/ftp_upload/61065/Supplemental_Figure_1.mp4" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about 3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry at JoVE.com

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

所提出的方法描述了如何识别和解决与二次离子质谱相关的测量伪影，以及如何获得固态材料中杂质/掺杂剂的真实 3D 分布。

使用二次离子质谱法重建分离杂质的 3D 深度剖面

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial ...

3d-depth-profile-reconstruction-segregated-impurities-using-secondary

Research

JoVE Journal

Chemistry

1.6K 次观看.  Łukasiewicz Research Network-Institute of Electronic Materials Technology. 所提出的方法描述了如何识别和解决与二次离子质谱相关的测量伪影，以及如何获得固态材料中杂质/掺杂剂的真实 3D 分布。 

视频: 3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

定量应用同质MALDI样品的制备

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

科研

生物化学

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Atom Probe Tomography Analysis of Exsolved Mineral Phases

Element diffusion rates and temperature/pressure control a range of fundamental volcanic and metamorphic processes. Such processes are often recorded in lamellae exsolved from host mineral phases. Thus, the analysis of the orientation, size, morphology, composition and spacing of exsolution lamellae is an area of active research in the geosciences. The conventional study of these lamellae has been conducted by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and more recently with focused ion beam (FIB)-based nanotomography, yet with limited chemical information. Here, we explore the use of atom probe tomography (APT) for the nanoscale analysis of ilmenite exsolution lamellae in igneous titanomagnetite from ash deposits erupted from the active Soufri&#232;re Hills Volcano (Montserrat, British West Indies). APT allows the precise calculation of interlamellar spacings (14&#8211;29 &#177; 2 nm) and reveals smooth diffusion profiles with no sharp phase boundaries during the exchange of Fe and Ti/O between the exsolved lamellae and the host crystal. Our results suggest that this novel approach permits nanoscale measurements of lamellae composition and interlamellar spacing that may provide a means to estimate the lava dome temperatures necessary to model extrusion rates and lava dome failure, both of which play a key role in volcanic hazard mitigation efforts.

Analysis of the morphology, composition, and spacing of exsolution lamellae can provide essential information to understand geological processes related to volcanism and metamorphism. We present a novel application of APT for the characterization of such lamellae and compare this approach to the conventional use of electron microscopy and FIB-based nanotomography.

原子探针断层扫描分析的解脱矿物相

Element diffusion rates and temperature/pressure control a range of fundamental volcanic and metamorphic processes. Such processes are often recorded in ...

环境科学

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.