Fig. 1: Spatial mass spectrometry techniques for molecular tissue imaging.

A Imaging Mass Cytometry (IMC) is a highly multiplexed imaging technique that uses metal-tagged antibodies (1) to detect over 40 protein markers simultaneously at subcellular resolution (~1 μm). A pulsed laser ablates the tissue at discrete locations (2), and the vaporized material is analyzed by time-of-flight mass spectrometry to identify the metal isotopes associated with each antibody. The image is constructed by raster-scanning the tissue and assigning the detected signals to spatial coordinates, generating high-dimensional maps where each pixel represents the abundance of specific protein markers (3). While IMC provides detailed spatial and phenotypic information about the tissue microenvironment, it is a destructive technique, meaning the same tissue section cannot be used for subsequent analyses, such as histological staining. B Mass Spectrometry Imaging (MSI) techniques, including Matrix-Assisted Laser Desorption/Ionization (MALDI) and Desorption Electrospray Ionization (DESI), enable label-free, untargeted imaging of a broad range of molecules, such as lipids, metabolites, and drugs, directly from tissue sections. MALDI-MSI requires matrix deposition (1) and uses a laser to desorb and ionize molecules from the coated surface (2), typically under vacuum, and can achieve high spatial resolution, down to 5–10 μm under optimized conditions. DESI-MSI operates under ambient conditions using a charged solvent spray to ionize molecules without the need for matrix application (2). This method involves minimal sample preparation and is compatible with in situ or near-real-time analysis. Depending on the specific configuration, DESI approaches can range from moderate to relatively high spatial resolution (<10 μm with nano-DESI MSI). MSI generates a three-dimensional data cube, where each x–y coordinate corresponds to a tissue location, and the third dimension represents the mass-to-charge (m/z) axis of the acquired spectrum. From this data, ion images can be reconstructed by selecting specific m/z values, producing spatial distribution maps of individual molecules. Each image reflects the localization and relative abundance of a given ion across the tissue, allowing direct correlation with morphological features (3). Importantly, MSI is generally minimally destructive, allowing subsequent histological staining (e.g., Haematoxilin and eosin staining) and enabling direct correlation between molecular and morphological features within the same tissue section.