Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.

质谱成像 (MSI) 使复杂表面上的分子分布可视化成为可能。它已广泛用于生物医学研究领域，以研究健康和患病组织。大多数 MSI 研究以 2D 方式进行，其中仅研究完整样本体积的单个切片。然而，生物过程发生在一个组织体积内，最好将其作为一个整体进行研究，以更全面地了解生物样品（如组织和细胞）的空间和分子复杂性。因此，质谱成像已扩展到 3D 领域，从而可以可视化 3D 样品中的分子分布。调查体积数据的好处导致单样本 3D-MSI 调查的应用迅速增加。要成功进行 3D-MSI 研究，需要考虑几个实验和数据分析方面。在这篇综述中，我们讨论了这些方面以及使 3D-MSI 能够常规应用于多样本研究的持续发展。