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质谱成像
质谱成像是一种分析技术，能够直观显示存在哪些分子以及它们所在的具体位置。其主要特点之一是能够同时对多种分子进行成像，有望在多个领域得到广泛应用。本页面为从事分析工作的人员介绍了质谱成像的原理、用途、注意事项以及样品制备相关基本技术信息。
滨松光子学株式会社开发了一款名为 Poropare 的样品制备产品，用于质谱成像中的转移采样。通过 Poropare，我们致力于让质谱成像技术变得更加触手可及、简单易用，并推动其在广泛的领域中得到应用。
质谱成像原理
质谱 (MS) 已经发展成为一项基础分析技术，它将基于分子质荷比 (m/z) 的卓越选择性与高探测效率相结合，能够对复杂样品中的众多组分进行同步分析。质谱成像 (MSI) 是一项创新技术，它利用质谱的特性，将组分的空间分布以图像形式呈现出来。这种空间信息通常无法通过需要进行样品提取的液相色谱-质谱 (LC/MS) 或气相色谱-质谱 (GC/MS) 获得，从而成为 MSI 相对于上述两种技术的一项显著优势。通常，MSI 分析按照以下步骤进行。
步骤 1：解吸与离子化
样品在局部完成解吸和离子化后，无需经过色谱分离，直接引入质谱仪，从而获得质谱图（即质荷比 (m/z) 与其强度的曲线图）。通过对整个样品进行这种局部过程的扫描，可获得一个包含每个像素点质谱图的数据集。
步骤 2：成像
数据通过专用 MSI 软件转换为图像。通过选择一个质荷比 m/z 并将其强度以色彩图谱的形式呈现出来，即可生成离子图像。对于每个选定的质荷比 m/z 值，都会生成一个不同的离子图像。
步骤 3：分析
将样品的光学图像与离子图像相结合，可以直观地分析目标离子（组分）被检测到的位置。
MSI 功能可作为质谱仪的一个选配项，所获得的结果取决于仪器规格。例如，如果一台仪器具有高质量分辨率，那么 MSI 数据也可以以同样高的质量分辨率进行采集。此外，MSI 通常能提供空间分辨率在几微米到几十微米范围内的离子图像（即分子分布图）。虽然影响分辨率的测量参数因方法而异，但在激光解吸/离子化方法中，激光照射直径和扫描间距会根据具体目的进行调整。
质谱成像的特点与挑战
特点
- 通过图像获得直观理解
离子图像以一种易于直观理解的形式呈现信息。解读质谱图对于非专业认识来说颇具挑战性，而离子图像则能够帮助简化理解。大面积成像（例如显微镜载玻片级别的成像）能够让人在接近肉眼可见的范围内，直观地了解组分分布情况。 - 多组分的同步成像
原则上，MSI 测量无需使用标记试剂来检测特定分子。此外，如上所述，由于每个像素点的质谱数据都存储下来，因此只需进行一次测量，即可实现多组分的成像。这有助于评估特定组分与其他组分之间的空间相关性。 - 用途多样
通过运用多种质谱技术，可以获得各类图像数据。例如，利用 MS/MS（串联质谱）或离子淌度技术进行痕量组分检测以及结构异构体分离，这些方法同样可应用于 MSI。此外，获得的离子图像可能会因所采用的离子化方法不同而有所差异。这些特点在多模态成像分析中非常有效。
挑战
- 初始成本高
除了质谱仪本身外，MSI 还需要样品制备设备（例如低温恒温器），这往往导致其初始成本高于其他成像技术。这也是阻碍 MSI 更广泛应用的因素之一。 - 分辨率与灵敏度之间的权衡
随着像素尺寸的减小，离子数量也往往随之减少，从而导致灵敏度下降。在优先考虑灵敏度的前提下设置灵敏度（通常约为 50 μm）时，可能难以将检测结果与显微镜或扫描电子显微镜 (SEM) 图像进行比较。近年来，通过提升质谱仪的性能和改进数据处理方法，人们一直在努力改善这一问题。 - 定量分析的局限性
由于 MSI 的特性——样品本身具有异质性，因此定量分析仍然面临挑战。样品表面的不规则性和组织特性共同造成的差异可能会影响信号强度。在解读信号强度以及比较不同样品之间的信号强度时，需格外谨慎。近年来，有一些能够考虑这种差异性的测量方案和分析方法相继提出。
MSI 采用的关键技术
MALDI
基质辅助激光解吸离子化 (MALDI) 是目前在 MSI 中使用最广泛的离子化方法。将一种有助于离子化的基质（即试剂）涂覆在样品薄切片的表面，然后使用紫外激光对其进行局部照射。该基质可吸收激光，并促进样品的解吸和离子化，从而在不产生显著碎裂的情况下实现离子化。MALDI 通常与飞行时间 (TOF) 质谱仪或四极杆飞行时间 (QTOF) 质谱仪结合使用。
DESI
解吸电喷雾离子化 (DESI) 是一种基于电喷雾离子化的技术，它通过喷射带电液滴来提取、解吸和离子化样品组分。它在许多基本方面与 MALDI 不同，并且正迅速成为一种备受欢迎的替代性 MSI 方式。虽然该技术通常与 QTOF 质谱仪结合使用，但最近也已实现了利用三重四极杆 (TQ) 质谱仪进行高灵敏度的靶向成像。
TOF-SIMS
飞行时间二次离子质谱 (TOF-SIMS) 通过向样品表面发射初级离子，并将溅射产生的二次离子引入飞行时间质谱仪 (TOF-MS) 来生成质谱图。该技术具有亚微米级空间分辨率和高表面灵敏度，是表面分析领域中广泛应用的一种技术。
尽管与 MALDI 和 DESI 相比，该技术往往会产生更多的离子碎裂，但近年来已经开发出能够实现软离子化的团簇离子源。
LA-ICP-MS
激光剥蚀电感耦合等离子体质谱 (LA-ICP-MS) 利用激光光束照射样品表面，并将剥蚀出的样品引入 ICP-MS 中。当样品在 ICP-MS 内被分解为原子状态时，即可获得各元素的图像。在生物科学领域，它用于分析生物样品中的微量金属元素。
MSI 所需的样品制备
薄切片
MSI 所需的样品制备步骤之一是制作薄切片。这指的是使用冷冻切片机将冷冻样品切成厚度约为 5 μm 至 20 μm 的切片的过程。这需要高超的技能，因为必须制备出厚度均匀的薄切片，同时还要避免导致样品组分的位移。
薄切片制备中的挑战示例
由于不同的样品具有易碎、坚硬或过薄等特性，薄切片制备可能会比较困难。由于不进行薄切片就无法进行测量，这一制备过程限制了 MSI 的应用范围。然而，通过采用可替代薄切片的替代采样方法（如转移采样）， MSI 的应用范围正在逐步扩大。
基质涂覆
使用 MALDI 进行 MSI 时，除了制备薄切片外，还需要进行基质涂覆。这个过程也需要专业知识和经验，例如选择适合样品的基质化合物，并将其均匀地涂覆在样品表面。此外，还有专门的基质涂覆设备可供使用，让这个过程变得更轻松。
我们致力于拓展 MSI 的应用范围
滨松光子学株式会社开发的 Poropare 是一种用于质谱成像的样品制备辅助产品，具有坚固的多孔结构。通过采用一种称为“转移取样”的工艺（该工艺充分利用了本产品的特性），可以在无需传统的样品薄切片制备过程的情况下进行 MSI。这使得 MSI 分析能够应用于更广泛的样品，包括那些此前因薄切片制备困难而无法使用 MSI 的样品。
此外，用于 MALDI 的 Poropare 在其板面预先涂覆了一层离子化辅助层，从而能够实现无基质分析。这大大简化了样品制备过程，降低了 MSI 的执行门槛，同时消除了基质引起的背景噪声，从而有助于提高信噪比 (S/N)，尤其是在低分子量化合物的分析中。另一方面，Poropare 也可与基质结合使用，以便分析在无基质条件下难以检测的组分。
*Poropare 是滨松光子学株式会社的注册商标。
相关用途
农作物的组分分析在作物品种以及农药和化肥的研发中起到极其重要的作用。阐明农作物中所含的化学组分及其生理功能，有望推动品种改良、农药和肥料的开发以及施用条件优化等领域的研究与发展，从而有助于提高农作物的产量和品质。为此，人们采用了多种分析方法，包括光谱法和质谱。除了分析仪器的开发外，提取和分离等样品制备流程近年来也日益精进，从而能够对痕量组分进行高灵敏度的检测和定量分析。
相关信息
质谱是一种用于识别和定量分析物质的方法，它通过各种离子化方法将物质转化为原子或分子级别的微小离子，进而测量其质荷比及数量。与其他分析方法相比，质谱在单次分析中能够提供更丰富的信息，因此可广泛应用于包括蛋白质组学在内的众多领域。
滨松光子学株式会社提供构成质谱仪核心的各种器件，包括离子检测器件。
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