Secondary Photoionisation Mass Spectrometry Imaging: The Physics Behind ‘Rescuing’ Signals from 99.9 per cent Neutral Molecules

If one were to summarise the value of secondary photoionisation (DESI) in mass spectrometry imaging with a single figure, it would be this: conventional DESI ionises less than 0.1 per cent of the desorbed molecules. The remaining 99.9 per cent or more are evacuated as neutral molecules and never enter the mass analyser.

This is not an exaggeration, but a simple physical calculation.

Estimates of the upper and lower limits of ionisation efficiency

A typical DESI desorption event releases approximately 1–10 pg (picograms) of material from the sample surface, comprising several million to tens of millions of molecules. These molecules are carried towards the mass spectrometer inlet in the form of aerosol droplets within the spray plume.

During the ESI ionisation process, the number of gas-phase ions that a charged droplet can produce in a Coulombic fragmentation sequence is constrained by the Rayleigh limit. As a simplified estimate: a charged droplet with an initial radius of approximately 1 μm can release around 100–1,000 gas-phase ions following complete evaporation and fragmentation. Calculated on the basis of a desorption quantity of 1 pg and an average molecular weight of 500 Da, the total number of molecules is approximately 1.2 × 10⁹. In other words, the ionisation fraction is approximately 10⁻⁷ to 10⁻⁶—that is, on the order of one part per million to one part per hundred thousand.

Even when the estimates are relaxed (larger droplets, more fragmentation cycles, more efficient desolvation), the ionisation efficiency of conventional ESI for neutral analytes rarely exceeds one thousandth. This is a physical limitation inherent to ESI itself, rather than an area for optimisation of operational parameters.

What does secondary photoionisation do?

The physical logic behind secondary photoionisation—or, more accurately, post-photoionisation—is simple: ESI performs the first round of ionisation, whilst VUV photons perform the second.

The key parameter is the photon flux of the VUV beam. A standard Kr discharge lamp (10.6 eV) generates a photon flux of approximately 10¹³–10¹⁴ photons/s/cm² in the ionisation zone. For a sampling area of 100 μm × 100 μm, the cross-sectional area of the ionisation zone is approximately 10⁻⁴ cm², resulting in the number of photons received per second being in the order of 10⁹–10¹⁰.

The time taken for a neutral molecule to traverse the ionisation region is of the order of microseconds (depending on the gas flow velocity); during this time, the probability of a molecule colliding with a photon depends on the photoionisation cross-section (σ, typically around 10⁻¹⁷–10⁻¹⁸ cm²). Under conditions of sufficiently high photon flux (the photon density of a Kr lamp can reach 10¹²–10¹³ photons/cm³), the probability of a neutral molecule being ionised whilst passing through the ionisation zone can reach a few per cent to several tens of per cent—compared to a few parts per million in pure ESI, this represents an improvement of 3–6 orders of magnitude.

This is the physical basis for the “1–3 orders of magnitude” signal gain achieved by secondary photoionisation mass spectrometry imaging (DESI). It is not a ‘black box’ parameter, but rather a physical process that can be quantitatively estimated from three parameters: photon flux, ionisation cross-section and molecular residence time.

How the two ionisation stages in MSI DPI work together

Neo Source INSTRUMENT’s MSI DPI imaging source positions the DESI spray and the VUV beam in spatial locations that are close to, but do not fully overlap. The rationale behind this design is as follows:

If the VUV beam were to completely cover the core ionisation region of the DESI spray, the ions generated by the initial ESI might be directly irradiated by VUV photons; high-energy photons could cause photodissociation, leading to a decrease in the intensity of molecular ion peaks and an increase in fragment peaks. MSI DPI positions the VUV beam downstream of the spray plume—at this point, most of the initial ESI ions have already entered the mass spectrometer inlet, whilst a large number of neutral molecules remain in the beam region due to their slower diffusion rate. This spatiotemporal separation ensures that the two ionisation processes do not interfere with one another: ESI handles polar molecules, whilst VUV is responsible for ‘pre-emptively’ intercepting molecules that are still in their neutral form.

Why is it referred to as “rescue”?

Secondary photoionisation does not alter the chemical mechanism of ESI, nor does it replace its function; it merely adds an ionisation pathway following ESI that does not rely on proton transfer. Describing it as “rescue” is more apt than “enhancement”—VUV photons do not help ESI perform better, but rather recover the 99.9 per cent of molecules that ESI is completely unable to reach.

In terms of the range of detectable species, secondary photoionisation expands the coverage of mass spectrometry imaging from ‘molecules easily ionised by ESI’ to ‘all molecules with an ionisation energy below 10.6 eV’. This expansion of the chemical space cannot be measured by numerical gains alone—many molecules that previously ‘did not exist’ in imaging have now begun to appear.

Related products: Neo Source INSTRUMENT MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source, spatial resolution 20–200 μm, compatible with Agilent, AB SCIEX and Thermo mass spectrometers; MSI LDPI Laser/Photochemical Ionisation Mass Spectrometry Imaging Source, spatial resolution 2–3 μm, also compatible with the three brands.