Tissue sections and H&E staining: how do molecular images align with the ‘pathological map’?

A tissue section is a standard sample preparation method in which biological tissue, following cryo- or paraffin-embedding, is cut into thin slices several micrometres thick and mounted onto a microscope slide for mass spectrometry imaging or microscopic observation. The thickness, flatness and quality of attachment of the section all affect imaging—sections that are too thick reduce resolution and impair desorption, whilst unevenness causes signal fluctuations. For mass spectrometry imaging, a good section forms the physical basis for obtaining a clear molecular distribution map.

H&E staining (hematoxylin and eosin staining) is the most commonly used histological staining method in pathology. Hematoxylin stains cell nuclei blue-violet, whilst eosin stains the cytoplasm and extracellular matrix pink; the combination of the two reveals cell morphology and tissue structure, serving as the reference image for pathologists to determine ‘which areas are pathological and which are normal’. It can be said that H&E images are ‘maps’ at the morphological level, whilst mass spectrometry imaging maps are ‘maps’ at the molecular level.

These two ‘maps’ must be superimposed upon one another to be diagnostically meaningful. Mass spectrometry imaging tells you ‘where a particular molecule is concentrated’, but it does not recognise tissue structure; H&E staining tells you ‘which part is tumour and which is normal’, but it cannot visualise molecules. Only by aligning the two on the same (or adjacent) section can one confirm ‘whether a particular marker is specifically enriched in the lesion’—which is precisely the evidence sought by molecular pathology.

Neo Source INSTRUMENT’s MSI DPI / MSI LDPI features a capability that aligns perfectly with this workflow: the imaging process involves atmospheric pressure and relatively gentle sampling, meaning the tissue section remains intact after imaging and can undergo further H&E histopathological examination, thereby enabling a ‘single section, molecular analysis followed by morphological analysis’ comparison. Case studies of melanocytic naevi on the official website demonstrate this comparison—by systematically comparing the imaging results of molecules such as cholesterol, S1P, PC34:1, PC38:4, and other molecules are compared one by one with the contours of the naevus area and normal tissue in the H&E images. It was found that the specific distribution of S1P, cholesterol and other molecules closely corresponds to the H&E naevus area, thereby confirming their value as distinguishing markers. Both sources can be fitted to mass spectrometers from Agilent, AB SCIEX and Thermo.