How mass spectrometry imaging works: how a tissue section is transformed into a ‘molecular map’

Imagine an ordinary histological section: under a microscope, you can see the morphology of the cells and the structure of the tissue, but you cannot tell where cholesterol is concentrated or where drug concentrations are high. Mass spectrometry imaging fills precisely this gap—it assigns a complete mass spectrum to each location, then represents the concentration of a particular molecule as a colour, ultimately producing a ‘molecular map’.

Below, we’ll explain how this works using the simplest possible steps.

How do ions ‘fly’ out?

The sample (such as a tissue section) is mounted on a stage, whilst the imaging source operates under ambient or near-ambient pressure conditions. Taking the imaging source from Neo Source INSTRUMENT as an example, the core technique is photoionisation: vacuum ultraviolet (VUV) photons are used to bombard molecules on the sample’s surface, causing them to lose electrons and become charged ions.

Some molecules are naturally prone to ionisation, whilst some neutral molecules require a ‘little extra push’. Post-photoionisation (secondary photoionisation) technology involves subjecting already vapourised neutral molecules to a second round of VUV photon irradiation, thereby ionising them. This step amplifies what would otherwise be a very weak signal by 1–3 orders of magnitude, allowing even low-abundance components to be detected.

How are positions ‘recorded’?

The ionised ions are drawn into the mass spectrometer. New Source’s ProC series of online time-of-flight mass spectrometers utilise a dual-field reflection TOF-MS configuration, in which ions are separated according to their mass-to-charge ratio (m/z) and recorded. Crucially, every time a spectrum is acquired, the system simultaneously records ‘the exact coordinates where the probe struck the sample at that moment’.

Consequently, each pixel is linked to a set of mass spectrometry data—revealing both “what molecules are present here” and “how many of these molecules there are”.

How is an image assembled?

The imaging control software directs the probe to move point by point (or line by line) across the sample surface, scanning the entire area much like a printer. Once the scan is complete, the software selects a specific m/z of interest to the user, extracts the signal intensity of that molecule from all pixels, and maps it to different shades of light and dark or cool and warm colours.

The same set of data can be ‘coloured’ repeatedly using different m/z values: one image shows lipid distribution, the next shows drug enrichment, and another shows metabolites. A single imaging session is equivalent to performing numerous traditional section staining procedures simultaneously, with the ‘molecular types’ rather than ‘morphology’ being highlighted.

Resolution determines how fine the details can be seen

The area covered by the probe at each stop corresponds to the size of a ‘pixel’. New Source’s MSI DPI covers 20–200 μm, making it suitable for examining large-scale distributions in tissues and materials; MSI LDPI reduces the pixel size to 2–3 μm, approaching the single-cell level, and can distinguish molecular differences between adjacent cells. Which one to choose depends on whether you wish to answer questions about ‘which area’ or ‘which cell’.

When broken down into its basic principles, mass spectrometry imaging is not mysterious: it involves charging molecules, recording their positions, and arranging them by colour. The real challenge lies in ensuring these three steps are stable and reproducible, whilst also being compatible with mainstream mass spectrometry platforms such as Agilent, AB SCIEX and Thermo—and this is precisely where the value of the imaging source as a standalone module lies.