Slice thickness and depth of field

Most mass spectrometry imaging studies utilise tissue sections, and the ‘thickness’ of these sections is a key experimental parameter affecting the results. In the life sciences, frozen sections are commonly used, typically with a thickness ranging from 10 to 20 μm: if the section is too thin, there are too few analyser molecules on the carrier, resulting in weak signals and high quantification noise; if too thick, the surface becomes uneven, making it difficult for the laser or spray to act uniformly; molecular signals from deeper layers are self-absorbed or quenched as they pass through, thereby reducing the effective signal. Thickness must therefore strike a balance between ‘sample loading’ and ‘signal penetration’.

‘Imaging depth’ refers to the depth range below the sample surface within which ions can still be effectively generated and detected. For atmospheric-pressure soft ionisation (DESI, LDPI), the process occurs primarily at the very surface, and it is often only the molecules within the top one or two micrometres that actually contribute to the signal; for hard ionisation or deep-penetration techniques (such as SIMS Secondary Ion Mass Spectrometry), depth information can be obtained through layer-by-layer sputtering. The ‘depth dimensions’ revealed by these two approaches are entirely different.

The relationship between thickness and lateral resolution is also asymmetrical. Lateral resolution is determined by the beam spot and step size (see Isotropy of spatial resolution and pixel step size), and can range from the sub-micrometre to the micrometre scale; whereas the ‘lateral resolution’ along the thickness direction is typically much coarser, limited by the slice thickness itself. Consequently, the information in the z-direction of a two-dimensional image is often an ‘average across the entire slice’; to obtain a true three-dimensional molecular distribution, it is necessary to cut multiple consecutive slices, image them separately, and then reconstruct the data.

Integration with the New Source workflow: Tissue sectioning is a standard pre-processing step for MSI imaging (see Tissue Sectioning and H&E Staining); whereas imprint imaging within the MSI family bypasses sectioning by directly imprinting the sample onto the target surface, thereby avoiding issues of longitudinal heterogeneity caused by thickness variations; this method is more suitable for scenarios where sectioning is not required, such as plants and thin-layer samples.

Practical advice: For comparative experiments within the same batch, the section thickness should be kept consistent; otherwise, signal intensities at different thicknesses cannot be directly compared, and quantitative conclusions may be distorted.

The type of section also affects the workflow: frozen sections are the mainstream method for MSI, as they preserve metabolites and small molecules well; paraffin-embedded sections, whilst convenient for long-term storage and pathological controls, require dewaxing and rehydration, and high-temperature embedding may result in the loss of volatile components; such losses must be assessed prior to using them for metabolite imaging. In contrast, blot imaging bypasses sectioning by directly transferring the sample onto the target surface, naturally avoiding longitudinal inhomogeneity along the thickness direction; it is therefore more suitable for scenarios where sectioning is unnecessary, such as plant leaves and thin-layer samples. Whichever method is used, maintaining consistent pre-processing is a prerequisite for comparability across samples.