Mass Spectrometry Imaging Ion Sources: Physical Evidence for the Elimination of Polarity Discrimination via Post-Photoionisation

Those working in mass spectrometry imaging all share the same experience: when imaging the same tissue section using conventional DESI, the signals from positively charged phospholipids (such as PC) are very strong, whilst neutral lipids such as sterols, fatty acids and triglycerides are barely detectable. This is not because these molecules are absent from the sample—when the same tissue homogenate is analysed by LC-MS/MS, the sterol content is not low. So where have the signals gone?

The problem lies in the ionisation stage.

Charge competition: a single ionisation site can only accommodate one ion

The basic process of electrospray ionisation (ESI) involves droplet evaporation, Coulombic fragmentation and the release of ions into the gas phase. During this process, all analytes to be ionised compete for charge sites on the droplet surface. The quaternary ammonium group (-N⁺(CH₃)₃) in phosphatidylcholine (PC) carries a permanent positive charge and has a very strong affinity for protons. When it coexists with cholesterol in the same droplet, PC virtually ‘monopolises’ all available protons, causing the cholesterol to become a neutral molecule in the electrospray plume—it is evaporated rather than ionised.

This is the classic “ion suppression effect”, which has been extensively discussed in the literature on liquid chromatography–mass spectrometry (LC–MS). In the context of mass spectrometry imaging, its fatal flaw lies in the fact that imaging involves a single desorption event without chromatographic separation; all co-desorbed molecules enter the ionisation region simultaneously, and ion suppression is determined entirely by the intrinsic ionisation efficiency of the molecules. Molecules with low ionisation efficiency become “invisible” from this stage onwards.

This behaviour is known as “polarity discrimination” – traditional ionisation sources yield high signals only for the molecules that are most readily ionised, whilst ignoring those with moderate or low ionisation efficiency.

The intervention point of post-photoionisation

Post-photoionisation has changed this situation.

In the workflow of the MSI DPI (Neo Source INSTRUMENT electrospray/photochemical ionisation mass spectrometry imaging source), desorption and ionisation are not carried out simultaneously, but are split into two separate steps:

1. DESI (Desorption and Electrospray Ionisation) – transfers molecules from the sample surface into the gas phase whilst generating a small number of initial ions;

1. VUV photoionisation — neutral molecules in the gas-phase plume are bombarded with vacuum ultraviolet photons (10.6 eV, Kr lamp) to ionise them directly.

Crucially, the physical mechanism of VUV photoionisation is entirely different from that of ESI. ESI relies on proton transfer/charge competition in the liquid phase, whereas VUV photoionisation relies on the matching of photon energy with the ionisation energy (IE) of the analyte.

A VUV photon carries 10.6 eV of energy. The ionisation energy of cholesterol is approximately 9.5–9.7 eV, that of fatty acids is approximately 9.8–10.2 eV, whilst that of PC-type phospholipids is approximately 8.5–9.0 eV. These molecules can all be ionised by a 10.6 eV photon—ionisation no longer depends on ‘who is better at snatching protons’, but rather on ‘which is more abundant in the gas-phase plume’.

Experimental evidence: a 50-fold signal increase for cholesterol

Direct comparative experiments on rat brain tissue sections illustrate this point most clearly.

In pure DESI mode, cholesterol (m/z 369.35, [M+H-H₂O]⁺) is barely visible in the imaging maps—the signal intensity is only slightly above the noise level. Upon switching to DESI+PI (with post-electron ionisation enabled), the signal intensity in the same m/z channel increased by approximately 50-fold, revealing a clear anatomical distribution in the white matter regions (where myelin is rich in cholesterol).

Signals from the PC class (m/z range 760–810) also showed a gain of approximately 3–5-fold—not because the ionisation efficiency of PC had improved, but because VUV photoionisation ‘recovered’ the portion of neutral PC molecules that had not been ionised during the first round of ESI.

The physical fact revealed by these data is that, in conventional DESI, more than 99.9 per cent of the molecules in a desorption spot’s molecular cloud remain in their neutral form—never having been ionised—before entering the mass spectrometer’s inlet. Post-ionisation essentially adds a separate, charge-competition-free ionisation step to the molecular cloud’s ‘exhaust stream’ —this is the physical source of the “signal gain of 1–3 orders of magnitude”.

Practical Implications

Polarity discrimination not only affects signal intensity but also the authenticity of the imaging results.

If, in an image, the PC channel appears bright whilst the cholesterol channel appears dark, this may represent a genuine concentration difference, or it may simply be an illusion created by ionisation discrimination. Post-photoionisation eliminates the interference caused by differences in ionisation efficiency on image contrast, allowing ion images to more closely reflect the true spatial distribution of molecules—the significance of this may extend beyond merely producing a ‘stronger signal’.

This has the most direct practical application in drug distribution imaging: the ionisation efficiency of drug molecules (particularly neutral drugs and metabolites) is often lower than that of endogenous lipids, meaning that traditional DESI images may underestimate—or even completely miss—the true drug concentration in target tissues. Post-electron ionisation provides a second ionisation pathway independent of ESI, which does not depend on the proton affinity of the drug molecule; any molecule with an ionisation energy below 10.6 eV can be detected.

Related products: Neo Source INSTRUMENT MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source, integrating DESI and PI dual modes, compatible with Agilent, AB SCIEX and Thermo mass spectrometers.