Principles of the secondary photoionisation mass spectrometry imaging source: a new approach to in situ imaging of non-polar metabolites

In mass spectrometry imaging, there is a class of molecules that have long posed a challenge for conventional ionisation methods: non-polar or weakly polar metabolites such as lipids, steroids and terpenes. Whilst these are highly endogenous and provide valuable information on their distribution, they are not easily and effectively captured by ionisation methods such as electrospray, which rely on charge transfer. Secondary photoionisation (post-photoionisation) tackles this problem by breaking it down into separate steps. This article explains the underlying principles and why this technique is well-suited to the in situ imaging of non-polar metabolites.

What is secondary photoionisation (post-ionisation)?

Secondary photoionisation, also known as post-ionisation, is based on the core concept of separating the processes of ‘desorption’ and ‘ionisation’. First, molecules on the sample surface are desorbed into the gas phase using electrospray ionisation (DESI) or a similar method; at this stage, most molecules are neutral. Next, a beam of photons (photochemical ionisation, PI) is directed at the desorbed gas-phase molecules, causing them to absorb the photon energy and become ionised. As ionisation occurs after desorption, this is termed ‘post-’ photoionisation.

The significance of this separation lies in the fact that desorption is responsible for ‘extracting the molecules’, whilst ionisation is responsible for ‘converting the molecules into ions’. Once the two processes are decoupled, the ionisation method is no longer constrained by the desorption process, allowing for the selection of more suitable photon energies for species that are difficult to ionise.

Why it is more effective for non-polar metabolites

Conventional DESI ionisation relies on liquid-phase charge transfer, which is highly efficient for polar and charged species but less so for non-polar species. Post-ionisation (PI) involves photons acting directly on gas-phase molecules, bypassing the preferences associated with charge transfer and making it more suitable for non-polar species such as lipids. Consequently, lipid signals that were previously weak or even absent in DESI spectra are significantly enhanced when the PI channel is activated.

The new Ceyuan MSI DPI’s DESI/PI dual-mode integrates these two pathways into a single source: DESI can be used for polar metabolites, whilst the PI channel can be activated for non-polar species such as lipids; a combination of both is also possible. Users can flexibly select the appropriate mode for the same sample, covering a wider range of molecular types.

The gain is quantifiable

Post-photoionisation does not merely result in a ‘perceived increase’ but delivers a measurable signal increment. In validated samples, enabling dual-mode operation increased the caffeine signal by approximately 61-fold (approximately 60 orders of magnitude), the theanine signal by approximately 14.5-fold, and the cholesterol signal in mouse brain by approximately 205-fold. The substantial increase in lipid signals such as cholesterol is a direct demonstration of the effective capture of non-polar species by secondary photoionisation.

In situ and matrix-free analysis are prerequisites for practical application

The value of the secondary photoionisation imaging source is only fully realised under ‘in situ, matrix-free’ conditions. MSI DPI operates under ambient pressure; samples do not require matrix spraying, thereby avoiding the suppression of signals at the low-mass end by matrix background and preserving the original distribution of the tissue sections. For precious clinical sections or irreplaceable samples, the matrix-free approach ensures that the samples are not chemically damaged, allowing for subsequent analysis such as HE staining.

Suitable Samples and Limitations

The post-photoionisation (DPI) scheme is suitable for studies focusing on the spatial distribution of non-polar metabolites such as lipids, steroids and terpenoids, as well as for broad-coverage imaging of mixed polar and non-polar samples. Its limitations are equally clear: spatial resolution is determined by the selected source (DPI of 20–200 μm); for high resolution at the cellular level, the 2–3 μm scheme of the MSI LDPI should be considered. The technology addresses ‘ionisation coverage’; resolution is a separate parameter for selection.

Summary

Secondary photoionisation, through its ‘desorption first, ionisation second’ separation process, has transformed the in situ imaging of non-polar metabolites from a challenge into a routine procedure. The dual-mode DESI/PI approach of New Source’s MSI DPI has demonstrated the effectiveness of this method on real samples with quantifiable gains (approximately 61-fold for caffeine and 205-fold for cholesterol).