Following from our previous Letter on this topic, this Article reports a detailed study of time-of-flight-secondary ion mass spectrometry (TOF-SIMS) positive ion spectra generated from a set of model biocompounds (arginine, trehalose, DPPC, and angiotensin II) by water cluster primary ion beams in comparison to argon cluster beams over a range of cluster sizes and energies. Sputter yield studies using argon and water beams on arginine and Irganox 1010 have confirmed that the sputter yields using water cluster beams lie on the same universal sputtering curve derived by Seah for argon cluster beams. Thus, increased ion yield using water cluster beams must arise from increased ionization. The spectra and positive ion signals observed using cluster beams in the size range from 1,000 to 10,000 and the energy range 5-20 keV are reported. It is confirmed that water cluster beams enhance proton related ionization over against argon beams to a significant degree such that enhanced detection sensitivities from 1 μm(2) in the region of 100 to 1,000 times relative to static SIMS analysis with Ar2000 cluster beams appear to be accessible. These new studies show that there is an unexpected complexity in the ionization enhancement phenomenon. Whereas optimum ion yields under argon cluster bombardment occur in the region of E/n ≥ 10 eV (where E is the beam energy and n the number of argon atoms in the cluster) and fall rapidly when E/n < 10 eV; for water cluster beams, ion yields increase significantly in this E/n regime (where n is the number of water molecules in the cluster) and peak for 20 keV beams at a cluster size of 7,000 or E/n ∼3 eV. This important result is explored further using D2O cluster beams that confirm that in this low E/n regime protonation does originate to a large extent from the water molecules. The results, encouraging in themselves, suggest that for both argon and water cluster beams, higher energy beams, e.g., 40 and 80 keV, would enable larger cluster sizes to be exploited with significant benefit for ion yield and hence analytical capability.

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C60 and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn(1) Val(5)]-angiotensin II and [Val(5)]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi3 (+), are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C60 ion beams are useful for peptide structural analysis.

RationaleTo discover the degree to which water‐containing cluster beams increase secondary ion yield and reduce the matrix effect in time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) imaging of biological tissue.MethodsThe positive SIMS ion yields from model compounds, mouse brain lipid extract and mouse brain tissue together with mouse brain images were compared using 20 keV C60 +, Ar2000 +, water‐doped Ar2000 + and pure (H2O)6000 + primary beams.ResultsWater‐containing cluster beams where the beam energy per nucleon (E/nucleon) ≈ 0.2 eV are optimum for enhancing ion yields dependent on protonation. Ion yield enhancements over those observed using Ar2000 + lie in the range 10 to >100 using the (H2O)6000 + beam, while with water‐doped (H2O)Ar2000 + they lie in the 4 to 10 range. The two water‐containing beams appear to be optimum for tissue imaging and show strong evidence of increasing yields from molecules that experience matrix suppression under other primary beams.ConclusionsThe application of water‐containing primary beams is suggested for biological SIMS imaging applications, particularly if the beam energy can be raised to 40 keV or higher to further increase ion yield and enhance spatial resolution to ≤1 µm. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

a Low ionization yields in time of flight secondary ion mass spectrometry (ToF-SIMS) particularly from single cells and tissues are proving to be a significant limitation in allowing this technique to reach its full potential. A number of approaches including embedding the sample in water or spraying water above sample surface has shown great prospective for increasing the ionization yield by a factor of 10 to 100 through 'proton mediated' reaction. Based on this hypothesis, a water cluster primary ion source has been developed in collaboration with Ionoptika Ltd to generate giant water cluster ions (H 2 O) n + (n = 1À10 000) using a similar supersonic jet expansion methodology as for argon cluster beams. The ion yields of arginine, cholesterol, angiotensin II and a lipid mix have been measured under static and high ion dose conditions using (H 2 O) 5000 + , (H 2 O) 3000 + , Ar 3000 + and C 60 + primary ion beams at 20 keV. An enhancement in yields up to a factor of around 4 is observed under water cluster impact, in comparison with C 60 + at 1 × 10 11 ions/cm 2 ion dose, whereas this increases by around 10-50 times at high ion dose conditions.

Time-of-f light SIMS is applied to the analysis of single cells and different types of biological tissue samples enabling the generation of images with high spatial resolution and chemical specificity. However, the low yield of secondary ions from this type of sample still remains a challenge. This low yield could potentially be increased by enhancing the protonation of ions with the presence of water. Here, we have explored the application of a prototype water cluster ion beam for the analysis of mouse brain tissue samples. A series of experiments acquired with 20 keV (H 2 O) 3000 + and 20 keV (H 2 O) 4500 + were compared with 20 keV C 60 + , showing ion yield enhancement when a (H 2 O) n + cluster ion is employed in the analysis. The results have demonstrated the potential benefits provided by the use of (H 2 O) n + clusters for the analysis of mouse brain tissue samples.