Mass spectrometry imaging answers where are what molecules, but looking only at the ion image often makes it difficult to judge the corresponding tissue structure (such as glands, interstitium, necrotic regions). H&E provides an authoritative morphological reference, enabling molecular enrichment to be localized to specific histological regions.
This collaboration is the basic paradigm of spatial biology: first obtain the molecular map by mass spectrometry, then annotate morphology by H&E, or register the two into a common coordinate framework, revealing the relationship between molecular heterogeneity and tissue partitioning.
H&E can be applied before or after mass spectrometry imaging, but attention must be paid to the effect of staining reagents on the mass spectrometry target surface and signals. A common practice is to perform mass spectrometry first on the same tissue (retaining molecular information), do H&E annotation on adjacent sections, or align via serial-section/mirror registration.
For matrix-free ambient sources (LDPI, DPI, DESI), imaging first then staining after sectioning is usually more direct; for MALDI, ensure the matrix and staining processes do not interfere with each other to avoid cross-contamination.
Registration relies on shared coordinates (position of the section on the slide, scale bar, landmarks). Section wrinkles and transfer displacement both reduce alignment accuracy, so standardization of sectioning and loading is a prerequisite for registration quality.
After registration, ion images and H&E can be overlaid in software (SCiLS Lab, MetaboScape), ROIs circled, and regional molecular spectra extracted to support regional-difference analysis.
H&E provides general morphology, and immunohistochemistry (IHC) provides localization of specific proteins; both can serve as morphological annotation layers for mass spectrometry imaging. For verification requiring single-molecule specificity, IHC supplements the deficiency of H&E, and the three form a multi-layer annotation system.
It is recommended to write serial sectioning + standard registration into the SOP to ensure comparability and reproducibility of the molecular map and morphological map.
To obtain detailed specifications, compatible models, or a quotation for the MSI LDPI / DPI full series imaging ion sources, visit the Neo-Source official website, or contact the official team for compatibility advice tailored to your mass spectrometer (Agilent / SCIEX / Thermo and other mainstream MS).