Quantitative and Qualitative Analyses of Mass Spectra of OEL Materials by Artificial Neural Network and Interface Evaluation: Results from a VAMAS Interlaboratory Study.

Quantitative analysis of binary mixtures of tris(2-phenylpyridinato)iridium(III) (Ir(ppy)3) and tris(8-hydroxyquinolinato)aluminum (Alq3) by using an artificial neural network (ANN) system to mass spectra was attempted based on the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study (TW2 A31) to evaluate matrix-effect correction and to investigate interface determination. Monolayers of binary mixtures having different Ir(ppy)3 ratios (0, 0.25, 0.50, 0.75, and 1.00), and the multilayers containing these mixtures and pure samples were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with different primary ion beams, OrbiSIMS (SIMS with both Orbitrap and ToF mass spectrometers), laser desorption ionization (LDI), desorption/ionization induced by neutral clusters (DINeC), and X-ray photoelectron spectroscopy (XPS). The mass spectra were analyzed using a simple ANN with one hidden layer. The Ir(ppy)3 ratios of the unknown samples and the interfaces of the multilayers were predicted using the simple ANN system, even though the mass spectra of binary mixtures exhibited matrix effects. The Ir(ppy)3 ratios at the interfaces indicated by the simple ANN were consistent with the XPS results and the ToF-SIMS depth profiles. The simple ANN system not only provided quantitative information on unknown samples, but also indicated important mass peaks related to each molecule in the samples without a priori information. The important mass peaks indicated by the simple ANN depended on the ionization process. The simple ANN results of the spectra sets obtained by a softer ionization method, such as LDI and DINeC, suggested large ions such as trimers. From the first step of the investigation to build an ANN model for evaluating mixture samples influenced by matrix effects, it was indicated that the simple ANN method is useful for obtaining candidate mass peaks for identification and for assuming mixture conditions that are helpful for further analysis.

Mass Spectrometry Imaging for Spatial Chemical Profiling of Vegetative Parts of Plants.

The detection of chemical species and understanding their respective localisations in tissues have important implications in plant science. The conventional methods for imaging spatial localisation of chemical species are often restricted by the number of species that can be identified and is mostly done in a targeted manner. Mass spectrometry imaging combines the ability of traditional mass spectrometry to detect numerous chemical species in a sample with their spatial localisation information by analysing the specimen in a 2D manner. This article details the popular mass spectrometry imaging methodologies which are widely pursued along with their respective sample preparation and the data analysis methods that are commonly used. We also review the advancements through the years in the usage of the technique for the spatial profiling of endogenous metabolites, detection of xenobiotic agrochemicals and disease detection in plants. As an actively pursued area of research, we also address the hurdles in the analysis of plant tissues, the future scopes and an integrated approach to analyse samples combining different mass spectrometry imaging methods to obtain the most information from a sample of interest.

Metabolism in action: stable isotope probing using vibrational spectroscopy and SIMS reveals kinetic and metabolic flux of key substrates.

Microbial communities play essential functions which drive various ecosystems supporting animal and aquatic life. However, linking bacteria with specific metabolic functions is difficult, since microbial communities consist of numerous and phylogenetically diverse microbes. Stable isotope probing (SIP) combined with single-cell tools has emerged as a novel culture-independent strategy for unravelling microbial metabolic roles and intertwined interactions in complex communities. In this study, we applied Raman and Fourier-transform infrared (FT-IR) spectroscopies, secondary ion mass spectrometry (SIMS) with SIP to probe the rate of 13C incorporation in Escherichia coli at 37 and 25 °C. Our results indicate quantitative enrichment and flow of 13C into E. coli at various time points. Multivariate and univariate analyses of Raman and FT-IR data demonstrated distinctive 13C concentration-dependent trends that were due to vibrational bands shifting to lower frequencies and these shifts were a result of incubation time and metabolic rate. SIMS results were in complete agreement with the spectroscopy findings, and confirmed the detected levels of 13C incorporation into microbial biomass at the investigated conditions. Having established that FT-IR and Raman spectroscopy with SIP can measure metabolism kinetics in this simple system, we have applied the kinetics concept to study the metabolism of phenol by Pseudomonas putida and metabolic interactions within a two-species consortia with E. coli that could not degrade phenol. Raman spectroscopy combined with SIP identified quantitative shifts in P. putida due to temporal assimilation of phenol. Although E. coli was unable to grow on phenol, in co-culture with P. putida, general metabolic probing using deuterated water for SIP revealed that E. coli displayed increasing metabolic activity, presumably due to cross feeding from metabolites generated by P. putida. This study clearly demonstrates that Raman and FT-IR combined with SIP provide rapid and sensitive detection of carbon incorporation rates and microbial interactions. These novel findings may guide the identification of primary substrate consumers in complex microbial communities in situ, which is a key step towards the characterisation of novel genes, enzymes and metabolic flux analysis in microbial consortia.

Microdistribution and quantification of the boron neutron capture therapy drug BPA in primary cell cultures of human glioblastoma tumour by NanoSIMS.

The ability of secondary ion mass spectrometry (SIMS) to provide high sensitivity imaging of elements and small-medium mass molecules in biological tissues and cells, makes it a very powerful tool for drug distribution studies. Here we report on the application of a high-resolution dynamic SIMS instrument for the quantification and localisation of therapeutic levels of the BNCT agent l-para-(dihydroxyboryl)-phenylalanine (BPA) in primary cell cultures from human patients exhibiting glioblastoma multiform tumours. Boron uptake and distribution was determined quantitatively as a function of cell-sampling location and different treatment regimes. Importantly, BPA was found to accumulate in cancer cells invading the 'brain around tumour' tissue in addition to the main tumour site. Pre-treatment of samples with l-tyrosine was found not to increase the uptake of BPA, nor change the intracellular drug distribution. In cultured cells from the tumour core and brain around tumour, with and without l-tyrosine pre-treatment, normalised boron-related signals were higher from cell nuclei than from cytoplasm. An efflux treatment was found to reduce BPA levels, but at a rate slower than the original uptake, and did not affect the intracellular drug distribution. To the best of our knowledge, these data represent the first published study of BPA uptake and l-amino acid pre-treatment in cultured primary human cells using dynamic SIMS, and the most detailed, subcellular distribution study of a BNCT agent in any cellular system.

The influence of polyatomic primary ion chemistry on matrix effects in secondary ion mass spectrometry analysis.

RATIONALE: The application of mass spectrometry imaging techniques to determine two- (2D) and three- (3D) dimensional chemical distribution ideally provides uniform, high sensitivity to multiple components and reliable quantification. These criteria are typically not met due to variations in sensitivity due to the chemistry of the analyte and surrounding surface chemistry. Here we explore the influence of projectile beam chemistry and sample chemistry in time-of-flight secondary ion mass spectrometry (TOF-SIMS). To the authors' knowledge this is the first time the combined effects of projectile chemistry and sample environment on the quantitative determination of mixed samples have been systematically studied. METHODS: Secondary ion yields of lipid and amino acid mixtures were measured under 20 keV C60 , Arn , and (H2 O)n cluster ion bombardment (n = 2000 or 4000) using TOF-SIMS. Ion suppression/enhancement effects were studied in dry sample films and in trehalose and water ice matrices. RESULTS: The extent of the matrix effects and the secondary ion yield were found to depend on the chemistry of the primary ion beam and (for C60 , Arn ) on the nature of the sample matrix. Under (H2 O)n bombardment the sample matrix had negligible effect on the analysis. CONCLUSIONS: Compared with C60 and Arn , water-containing cluster projectiles enhanced the sensitivity of TOF-SIMS determination of the chosen analytes and reduced the effect of signal suppression/enhancement in multicomponent samples and in different sample matrices. One possible explanation for this is that the (H2 O)4000 projectile initiates on impact a nanoscale matrix environment that is very similar to that in frozen-hydrated samples in terms of the resulting ionisation effects. The competition between analytes for protons and the effect of the sample matrix are reduced with water-containing cluster projectiles. These chemically reactive projectile beams have improved characteristics for quantitative chemical imaging by TOF-SIMS compared with their non-reactive counterparts.

Quartz Crystal Microbalance Assay of Clinical Calcinosis Samples and Their Synthetic Models Differentiates the Efficacy of Chelation-Based Treatments.

This paper sets out in vitro protocols for studying the relative effectiveness of chelators used in the dissolution-based treatment of hard calcinosis. Pulverized hard calcinosis samples from human donors or synthetic hydroxyapatite nanoparticles were deposited by electrophoretic deposition on the surface of a quartz crystal microbalance sensor. Over 150 deposits of <20 µg were dissolved over the course of 1 h by aliquots of buffered, aqueous solutions of two calcium chelators, EDTA and citrate, with the surface-limited dissolution kinetics monitored with <1 s time resolution. There was no statistically significant difference in dissolution rate between the four synthetic hydroxyapatite materials in EDTA, but the dissolution rates in citrate were lower for hydroxyapatite produced by acetate or nitrate metathesis. Hard calcinosis and synthetic hydroxyapatites showed statistically identical dissolution behavior, meaning that readily available synthetic mimics can replace the rarer samples of biological origin in the development of calcinosis treatments. EDTA dissolved the hydroxyapatite deposits more than twice as fast as citrate at pH 7.4 and 37 °C, based on a first-order kinetic analysis of the initial frequency response. EDTA chelated 6.5 times more calcium than an equivalent number of moles of citrate. Negative controls using nonchelating N,N,N',N'-tetraethylethylenediamine (TEEDA) showed no dissolution effect. Pharmaceutical dissolution testing of synthetic hydroxyapatite tablets over 6 h showed that EDTA dissolved the tablets four to nine times more quickly than citrate.

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques.

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways.

Peptide Fragmentation and Surface Structural Analysis by Means of ToF-SIMS Using Large Cluster Ion Sources.

Peptide or protein structural analysis is crucial for the evaluation of biochips and biodevices, therefore an analytical technique with the ability to detect and identify protein and peptide species directly from surfaces with high lateral resolution is required. In this report, the efficacy of ToF-SIMS to analyze and identify proteins directly from surfaces is evaluated. Although the physics governing the SIMS bombardment process precludes the ability for researchers to detect intact protein or larger peptides of greater than a few thousand mass unit directly, it is possible to obtain information on the partial structures of peptides or proteins using low energy per atom argon cluster ion beams. Large cluster ion beams, such as Ar clusters and C60 ion beams, produce spectra similar to those generated by tandem MS. The SIMS bombardment process also produces peptide fragment ions not detected by conventional MS/MS techniques. In order to clarify appropriate measurement conditions for peptide structural analysis, peptide fragmentation dependency on the energy of a primary ion beam and ToF-SIMS specific fragment ions are evaluated. It was found that the energy range approximately 6 ≤ E/n ≤ 10 eV/atom is most effective for peptide analysis based on peptide fragments and [M + H] ions. We also observed the cleaving of side chain moieties at extremely low-energy E/n ≤ 4 eV/atom.

Matrix effects in biological SIMS using cluster ion beams of different chemical composition.

The influence of the matrix effect on secondary ion yield presents a very significant challenge in quantitative secondary ion mass spectrometry (SIMS) analysis, for example, in determining the relative concentrations of metabolites that characterize normal biological activities or disease progression. Not only the sample itself but also the choice of primary ion beam may influence the extent of ionization suppression/enhancement due to the local chemical environment. In this study, an assessment of ionization matrix effects was carried out on model systems using C60 (+), Arn (+), and (H2O)n (+) cluster ion beams. The analytes are pure and binary mixtures of amino acids arginine and histidine biological standards. Ion beams of 20 keV were compared with a range of cluster sizes n = 1000-10 000. The component secondary ion yields were assessed for matrix effects using different primary ion beams and sample composition. The presence of water in the cluster beam is associated with a reduction in the observed matrix effects, suggesting that chemically reactive ion beams may provide a route to more quantitative SIMS analysis of complex biological systems.

Evaluation of biomolecular distributions in rat brain tissues by means of ToF-SIMS using a continuous beam of Ar clusters.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides detailed chemical structure information and high spatial resolution images. Therefore, ToF-SIMS is useful for studying biological phenomena such as ischemia. In this study, in order to evaluate cerebral microinfarction, the distribution of biomolecules generated by ischemia was measured with ToF-SIMS. ToF-SIMS data sets were analyzed by means of multivariate analysis for interpreting complex samples containing unknown information and to obtain biomolecular mapping indicated by fragment ions from the target biomolecules. Using conventional ToF-SIMS (primary ion source: Bi cluster ion), it is difficult to detect secondary ions beyond approximately 1000 u. Moreover, the intensity of secondary ions related to biomolecules is not always high enough for imaging because of low concentration even if the masses are lower than 1000 u. However, for the observation of biomolecular distributions in tissues, it is important to detect low amounts of biological molecules from a particular area of tissue. Rat brain tissue samples were measured with ToF-SIMS (J105, Ionoptika, Ltd., Chandlers Ford, UK), using a continuous beam of Ar clusters as a primary ion source. ToF-SIMS with Ar clusters efficiently detects secondary ions related to biomolecules and larger molecules. Molecules detected by ToF-SIMS were examined by analyzing ToF-SIMS data using multivariate analysis. Microspheres (45 µm diameter) were injected into the rat unilateral internal carotid artery (MS rat) to cause cerebral microinfarction. The rat brain was sliced and then measured with ToF-SIMS. The brain samples of a normal rat and the MS rat were examined to find specific secondary ions related to important biomolecules, and then the difference between them was investigated. Finally, specific secondary ions were found around vessels incorporating microspheres in the MS rat. The results suggest that important biomolecules related to cerebral microinfarction can be detected by ToF-SIMS.

Mass spectrometric imaging of brain tissue by time-of-flight secondary ion mass spectrometry--How do polyatomic primary beams C60⁺, Ar2000⁺, water-doped Ar2000⁺ and (H2O)6000⁺ compare?

RATIONALE: To 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. METHODS: The 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. RESULTS: Water-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 (H2 O)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. CONCLUSIONS: The 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.

Enhancing ion yields in time-of-flight-secondary ion mass spectrometry: a comparative study of argon and water cluster primary beams.

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.

Spatiotemporal lipid profiling during early embryo development of Xenopus laevis using dynamic ToF-SIMS imaging.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used for the direct analysis of single intact Xenopus laevis embryo surfaces, locating multiple lipids during fertilization and the early embryo development stages with subcellular lateral resolution (∼4 µm). The method avoids the complicated sample preparation for lipid analysis of the embryos, which requires selective chemical extraction of a pool of samples and chromatographic separation, while preserving the spatial distribution of biological species. The results show ToF-SIMS is capable of profiling multiple components (e.g., glycerophosphocholine, SM, cholesterol, vitamin E, diacylglycerol, and triacylglycerol) in a single X. laevis embryo. We observe lipid remodeling during fertilization and early embryo development via time course sampling. The study also reveals the lipid distribution on the gamete fusion site. The methodology used in the study opens the possibility of studying developmental biology using high resolution imaging MS and of understanding the functional role of the biological molecules.

Quantitative surface analysis of a binary drug mixture--suppression effects in the detection of sputtered ions and post-ionized neutrals.

A systematic mass spectrometric study of two of the most common analgesic drugs, paracetamol and ibuprofen, is reported. The drugs were studied by means of secondary ion mass spectrometry (SIMS) and secondary neutral mass spectrometry (SNMS) using laser post-ionization (LPI) both in pure samples and in a two-component mixture. Ion suppression within the two-component system observed in SIMS mode is ameliorated using LPI under room temperature analysis. However, suppression effects are apparent in LPI mode on performing the analysis at cryogenic temperatures, which we attribute to changes in the desorption characteristics of sputtered molecules, which influences the subsequent post-ionization efficiency. This suggests different mechanisms of ion suppression in SIMS and LPI modes.

Secondary ion mass spectrometry imaging of biological cells and tissues.

Secondary ion mass spectrometry (SIMS) is capable of providing detailed atomic and molecular characterization of the surface chemistry of (bio)molecular samples. It is one of a range of mass spectrometry imaging techniques that combine the high sensitivity and specificity of mass spectrometry with the capability to view the distribution of analytes within solid samples. The technique is particularly suited to the detection and imaging of small molecules such as lipids and other metabolites. A limit of detection in the ppm range and spatial resolution <1 µm can be obtained. Recent progress in instrumental developments, including new cluster ion beams, the implementation of tandem mass spectrometry (MS/MS), and the application of multivariate data analysis protocols promise further advances. This chapter presents a brief overview of the technique and methodology of SIMS using exemplar studies of biological cells and tissue.

Peptide structural analysis using continuous Ar cluster and C60 ion beams.

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.

Enhancing secondary ion yields in time of flight-secondary ion mass spectrometry using water cluster primary beams.

Low secondary ion yields from organic and biological molecules are the principal limitation on the future exploitation of time of flight-secondary ion mass spectrometry (TOF-SIMS) as a surface and materials analysis technique. On the basis of the hypothesis that increasing the density of water related fragments in the ion impact zone would enhance proton mediated reactions, a prototype water cluster ion beam has been developed using supersonic jet expansion methodologies that enable ion yields using a 10 keV (H2O)1000(+) beam to be compared with those obtained using a 10 keV Ar1000(+) beam. The ion yields from four standard compounds, arginine, haloperidol, DPPC, and angiotensin II, have been measured under static+ and high ion dose conditions. Ion yield enhancements relative to the argon beam on the order of 10 or more have been observed for all the compounds such that the molecular ion yield per a 1 µm pixel can be as high as 20, relative to 0.05 under an argon beam. The water beam has also been shown to partially lift the matrix effect in a 1:10 mixture of haloperidol and dipalmitoylphosphatidylcholine (DPPC) that suppresses the haloperidol signal. These results provide encouragement that further developments of the water cluster beam to higher energies and larger cluster sizes will provide the ion yield enhancements necessary for the future development of TOF-SIMS.

TOF-SIMS with argon gas cluster ion beams: a comparison with C60+.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.

Three-dimensional mass spectral imaging of HeLa-M cells--sample preparation, data interpretation and visualisation.

Time-of-flight secondary ion mass spectrometry (ToFSIMS) is being applied increasingly to the study of biological systems where the chemical specificity of mass spectrometry and the high lateral resolution imaging capabilities can be exploited. Here we report a comparison of two cell sample preparation methods and demonstrate how they influence the outcome of the ToFSIMS analysis for three-dimensional (3D) imaging of biological cells using our novel buncher-ToF instrument (J105 3D Chemical Imager) equipped with a C(60) primary ion beam. Cells were analysed fixed and freeze-dried and non-fixed, frozen-hydrated. It is concluded that maintaining the cells in a non-fixed frozen-hydrated state during the analysis helps reduce chemical redistribution, producing cleaner spectra and improved chemical contrast in both 2D and 3D imaging. Insights into data interpretation are included and we present methods for 3D reconstruction of the data using multivariate analysis techniques.