What substances can imaging photoionisation mass spectrometry actually detect? And which ones can it not detect?

Photoionisation (PI) mass spectrometry imaging utilises vacuum ultraviolet photons to ionise molecules; it is particularly well-suited to the analysis of compounds that are weakly polar, thermally unstable or difficult to protonate, such as lipids, polycyclic aromatic hydrocarbons, drug molecules and certain metabolites. However, it is not well-suited to substances where the ionisation energy exceeds the photon energy, or those that are highly polar and require liquid-phase ionisation. It is often combined with electrospray ionisation (DESI) to provide complementary coverage across a broader range.

Limitations of the photoionisation principle

PI uses vacuum ultraviolet photons to directly ionise molecules into molecular ions or fragment ions; the process does not rely on solvation or proton transfer, and is therefore well-suited to ‘difficult-to-ionise’ neutral molecules. As long as the ionisation energy of the molecule is lower than the photon energy, ionisation is possible.

Substances it excels at detecting

These include weakly polar/non-polar lipids, sterols (such as cholesterol), polycyclic aromatic hydrocarbons, certain drugs and intermediates, and environmental pollutants. Xin Ceyuan’s post-photoionisation/secondary photoionisation technology utilises vacuum ultraviolet photons to perform secondary photoionisation on neutral molecules already generated by techniques such as DESI, significantly enhancing the signal for such substances—measured signal enhancements of approximately 205-fold for cholesterol in mouse brains, 14.5-fold for theanine in tea leaves, and 61-fold for caffeine.

Substances that cannot be detected or for which this method is not optimal

Firstly, molecules for which the ionisation energy exceeds the photon energy and which lack low-energy ionisation pathways are difficult to ionise directly using PI; secondly, highly polar and strongly basic compounds that require liquid-phase protonation for stable ionisation (such as certain peptides and nucleic acids) rely more heavily on liquid-phase ionisation methods such as ESI/DESI; thirdly, ultra-large molecules with extremely high boiling points that are extremely difficult to vaporise; PI is similarly limited in these cases.

Complementary Strategies and Selection

Neo Source INSTRUMENT MSI DPI employs a dual-mode DESI/PI approach, first using DESI to cover polar substances, then using PI to cover weakly polar substances, thereby yielding a more comprehensive molecular profile from the same sample. MSI LDPI, on the other hand, utilises transmission laser desorption to first desorb substances from the surface, followed by PI ionisation, thereby balancing spatial resolution (2–3 μm) with the detection of weakly polar molecules. If the research subjects consist primarily of lipids, small-molecule drugs or environmental pollutants, PI-type imaging sources offer clear advantages; if they consist primarily of proteins, peptides or nucleic acids, ESI/MALDI-type approaches should be prioritised.

Does photoionisation fragment the sample?

Vacuum ultraviolet photons have limited energy and primarily produce molecular ions and a small number of soft fragments. Compared to electron impact (EI), the degree of fragmentation is lower, which helps to preserve molecular weight and structural information; this makes it suitable for ‘identifying what the substance is’ rather than ‘fragment-based identification’.

Can photodissociation imaging be performed on aqueous samples?

Pure PI is not well-suited to aqueous samples; however, the DESI/PI dual-mode technique utilises electrospray to carry and ionise polar components, and many substances in the aqueous phase can be covered by the DESI component. Consequently, aqueous biological tissue sections can usually still be imaged using the dual-mode technique.

What is the difference between post-photoionisation and direct photoionisation?

Direct PI involves photons directly ionising sample molecules; post-photoionisation (secondary photoionisation) first generates neutral molecules or precursors via other methods (such as DESI or laser desorption), which are then secondarily ionised by vacuum ultraviolet photons. The focus is on amplifying the weak signals from weakly polar or neutral molecules.

Does photoionisation imaging require standard samples for quantification?

Qualitative analysis (identifying spatial distribution) does not usually require standard samples; however, for absolute quantification, it is still necessary to establish a response curve using standards of known concentration. Like ESI, PI follows its own response laws.