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Traditional solution and solid state approaches (Nuclear Magnetic Resonance, X-Ray Crystallography) are methods of choice when analysing both biological and inorganic analytes. However, the characterisation of transient species, often encountered in self-assembling systems, is difficult. Such systems rarely produce crystals of high quality and due to their dynamic nature; their structures are difficult to study with NMR. Hyphenated gas phase methods which rely on mass spectrometry detection offer simultaneous structural analysis and direct stoichiometry measurement. As a consequence, it is possible to investigate specific, non-interacting molecules and molecular complexes in an isolated environment. This thesis focuses on the development and applications of two such methods – ion mobility mass spectrometry (IM-MS) and cold ion spectroscopy. IM-MS measurements yield a so called collisional cross sectional area (CCS). This parameter can be pictured as a rotationally averaged, shadow projection of a molecule structure. When correlated with the ion abundance, a CCS distribution yields intuitively interpretable information about the conformational preferences of an isolated molecule. Although indispensable in describing a “global” geometrical structure, the CCS parameter itself provides a limited insight into the local structural features of the assembly. Ion spectroscopy, both in the UV and IR regions, can provide an extra layer of highly descriptive information. Here, we present several cases where the above techniques have been applied. With the aid of IM-MS, we have analysed the geometry of inorganic supramolecular assemblies, highlighting the stability of particular metal-ligand interactions. Using cold ion spectroscopy, we have assessed the fine structural information of self-assembled oligomers of an amyloidogenic peptide. We correlated spectral features of isolated oligomers to features observed in the mature fibrils; therefore attempting to delineate the events in early stages of amyloidogenic aggregation. A major part of this report focusses on technological aspects of the design and development of a high resolution, variable temperature ion mobility mass spectrometer (VT-IM-MS). The thermal stability of molecules is a vital aspect in industrial process development and formulation science. Solution phase Differential Scanning Calorimetry (DSC) is a widely applied technique, allowing to monitor reversibility of thermally induced conformational transitions, a key aspect in protein folding analysis. The instrument reported here aims to provide parallel information about gaseous ions, with a particular focus on protein ions. Capabilities of the newly built instrument have been tested using small, rigid molecules, a small protein and a large multiprotein complex.

Secondary ion mass spectrometry excels at determining the spatialdistribution of lipids in tissue, and thus has been used as a tool to elucidate thepathophysiologic mechanisms/etiology of Alzheimer’s disease. However, secondaryion mass spectrometry struggles to detect larger macromolecules such as thecharacteristic protein plaques associated with Alzheimer’s disease. This project wasundertaken to determine a protocol that would identify Alzheimer’s amyloid plaquesusing secondary ion mass spectrometry and then compare the plaque location to thelipid distribution within the diseased brain tissue. To accomplish this, samples werestained using Thioflavin T, a fluorescent molecule whose size is within theinstrument's range of detection and that specifically binds to amyloid plaques. Thesamples were then analysed with secondary ion mass spectrometry to detect thecharacteristic fragment of Thioflavin T. This method proved successful and allowedfor the comparison of amyloid plaque locations with the spatial distribution ofvarious lipid species. Using secondary ion mass spectroscopy provides unique spatial determination of lipids and molecules in tissue samples, yet this technique is unable to identify larger proteins. Thus, in order to identify the very large, hallmark plaques of Alzheimer's disease, a novel approach had to be developed. By using a small-molecular-weight molecule that binds to these hallmark plaques, we were able to identify where in the tissue these plaques were located using Secondary Ion Mass Spectroscopy.

The University of ManchesterFaculty of Engineering and Physical SciencesAbstract of thesis entitled ‘Systems Biology of HIF Metabolism in Cancer’Submitted by Emily Grace Armitage for the degree of Doctor of Philosophy, September 2012Cancer is one of the most devastating human diseases that cause a vast number of mortalities worldwide each year. Cancer research is one of the largest fields in the life sciences and despite many astounding breakthroughs and contributions over the past few decades, there is still a considerable amount to unveil on the function of cancer that would improve diagnostics, prognostics and therapy. Since cancer is known to involve a wide range of processes, applying methods to study it from a systems perspective could reveal new properties of cancer. Systems biology is becoming an increasingly popular tool in the life sciences. The approach has been applied to many biological and biomedical analyses drawing upon recent advancements in technology that make high throughput analyses of samples and computational modelling possible. In this thesis, the effect of hypoxia inducible factor-1 (HIF-1) on cancer metabolism, the entity considered most closely related to phenotype has been investigated. This transcription factor is known to regulate a multitude of genes and proteins to promote survival in a low oxygen environment that is prevalent in solid tumours. However its effect on the metabolome is less well characterised. By revealing the effect of HIF-1 on the metabolome as a system it is hoped that phenotypic signatures, key metabolic pathways indicative of cancer function and potential targets for future cancer therapy, can be revealed.The system has been studied using two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby metabolism has been profiled using a range of analytical platforms. In each model, wild type cells have been compared to cells deficient in HIF-1 to reveal its effect on cellular metabolism. Gas chromatography mass spectrometry (GC MS) and ultra high performance liquid chromatography - mass spectrometry (UHPLC MS) have been employed for metabolic profiling of cells exposed to a range of oxygen conditions. Additionally, time-of-flight secondary ion mass spectrometry (ToF SIMS) has been employed for imaging mass spectrometric analysis of multicellular tumour spheroids cultured from wild type cells and cells with dysfunctional HIF-1 to represent small initiating tumours. Using these techniques in metabolic profiling it has been possible to reveal metabolites associated with the effect of oxygen and HIF-1 on cancer metabolism along with key pathways and hubs that could be targeted in future therapy. Using imaging mass spectrometry it has been possible to localise metabolites in situ revealing how tumour structure relates to function. Finally, a novel approach to consider how metabolites are correlated with one another in the response to oxygen level or presence or absence of functional HIF-1 has been undertaken to better understand the systems properties of cancer metabolism. Metabolites found to be differently correlated with respect to oxygen and/or HIF-1 have been mapped onto a human metabolic network to determine their network-based origins. This allowed the simulation of sub-networks of metabolism most affected by oxygen and HIF-1, highlighting the key mechanisms in HIF 1 mediated cancer cell survival. Lay AbstractThe University of ManchesterFaculty of Engineering and Physical SciencesLay Abstract of thesis entitled ‘Systems Biology of HIF Metabolism in Cancer’Submitted by Emily Grace Armitage for the degree of Doctor of Philosophy, September 2012Cancer is one of the most devastating human diseases that cause a vast number of mortalities worldwide each year. Cancer research is one of the largest fields in the life sciences and despite many astounding breakthroughs and contributions over the past few decades, there is still a considerable amount to unveil on the function of cancer that would improve diagnostics, prognostics and therapy. Since cancer is known to involve a wide range of processes, applying methods to study it as a whole system could reveal new properties of cancer. Systems biology is becoming an increasingly popular tool in the life sciences. The approach has been applied to many biological and biomedical analyses drawing upon recent advancements in technology. When tumours grow, their oxygen supply becomes scarce and they need to employ mechanisms to overcome this for survival. In this thesis, tumour metabolism has been investigated with particular emphasis on how tumours respond to low oxygen. This has involved the analysis small molecules using state of the art techniques to determine which molecules are caused by and appear to promote survival in tumour cells in low oxygen. These molecules have also been imaged in cross sections of tumours to see where they occur and to hypothesise the function of these molecules based on their position with respect to oxygen availability. Using the data from each analysis it has been possible to link together knowledge about the molecules revealed to consider how tumours survive in low oxygen as a whole system.

In this study, the components of a biotechnologically relevant E. coli expressionsystem were characterised through the use of mass spectrometry techniques. Theseinclude the bacterial host itself, the enzymatic product of the P450 RhF geneexpression which catalyses a selective hydroxylation reaction that produces 5-hydroxydiclofenac from a diclofenac substrate; and both the substrate and productof the reaction. The identification of molecular signals as phosphatidylglicerol (PG),phosphatidylethanolamine (PE) and fatty acids (FA) led to the generation of a lipidprofile. Instrumentation relying on different ionisation mechanisms was exploredand bioinformatic tools were used to extract useful information from large imagingdata sets through normalisation and PCA analysis.

The 21st Century has seen a rise in incidence of complex diseases such as cancer and in the quest to develop essential new therapeutic options, the study of drug-cell interactions can yield powerful information. Acute myeloid leukaemia (AML) is an aggressive cancer that causes life-threatening deficits of functional blood cells in humans for which current treatment options are highly toxic and often poorly tolerated. A combination of two existing drugs, bezafibrate and medroxyprogesterone acetate in a drug redeployment situation has shown promise in vitro and in vivo and further investigations are crucial to elucidate the mode of action of this treatment.This project investigated the mechanistic action of BaP at a cellular level. Orthogonal spectroscopic and mass spectrometric platforms were employed to probe the biochemical composition of two AML cell lines, HL60 and K562 in the presence and absence of this combined drug treatment. Analysis was performed on single living cells, dehydrated cells, fixed cells and cell extracts to give a large and detailed data set.A consideration of the main spectral differences obtained by Synchrotron-FTIR and ATR-FTIR in conjunction with multivariate statistical analysis revealed a significant change to the cellular lipid composition with drug treatment; furthermore, this response was not caused by cell apoptosis. In particular, the ratio of CH2:CH3 was observed to increase with BaP treatment and this was determined to be a significant change in both cell lines (p < 0.05). An overall increase in lipid unsaturation suggests that BaP targets cellular lipid biosynthesis. Raman microspectroscopy added a further dimension to the spectroscopic study by providing spatial information of lipid distribution which suggested that BaP-induced saturation change is uniform across a single cell.UHPLC-MS was employed for global metabolomics analysis of AML cell extracts and revealed a number of biochemical pathways that were indicated as targets of BaP therapy in both cell lines. Univariate and multivariate analysis determined statistically significant metabolites for which putative identifications were made. Pyrimidine metabolism was the most significant pathway identified for changes consistent in both HL60 and K562 cell lines.The complementarity of ToF-SIMS and UHPLC-MS provided large coverage of the lipidome of AML cells through untargeted and targeted approaches. For data derived by both techniques, a general increase in polyunsaturated species for BaP treated cell extracts was observed which correlated well with findings from spectroscopic investigations.Adopting a multi-disciplinary approach to cell analysis can afford a powerful insight into understanding drug mode of action at a cellular level and novel information regarding BaP mechanistic action in AML cell lines was revealed. This analytical approach could be extended to the future study of drug-cell interactions for other oncological systems.

ToF-SIMS imaging has been drawing attention due to the wide range of applications in the biological and biomedical fields. These applications include the acquisition of quantitative and qualitative data that ranges in scale from single cells to organs, image visualisation and interpretation of biomarkers for diagnosis and development of pharmaceutics. This study focused on molecular imaging of mouse brain tissue sections using cluster primary ion beams. First, cluster ion beams were applied to comparative background studies of biomolecules and brain total lipid extract. Enhancement of the secondary ion signal was observed using water-containing cluster primary ion beams, especially for [M+H]+ type secondary ions. Water-containing clusters were then used to acquire ToF-SIMS images from the cerebellar area of serial mouse brain tissue sections. Again, water-containing cluster beams produced the highest secondary ion yields in both grey and white matter, gaining a new level of insight into the lipid compositions of both types of tissue in the brain. A clinical case was also evaluated with ToF-SIMS imaging, using cluster beams for the analysis of 3xTg-AD mouse brain tissue. SIMS images were registered with fluorescence microscopy images for the in situ identification and co-localisation of the Amyloid-β plaques on the SIMS images. Spectra from regions of interest were analysed to identify possible ion fragments derived from the Aβ protein. The co-localisation of cholesterol was also studied from images obtained with different primary ion beams. The results presented show that cluster ToF-SIMS can be successfully applied to brain tissue imaging. New primary ion beam technologies allow us to acquire data with more useful secondary ion yield for clinical applications and biological research. Nevertheless, future technological improvements are required for specialised applications e.g. cellular imaging. Moreover, processing the data obtained is still challenging and more data processing tools are also needed for interpretation.

High surface sensitivity and lateral resolution imaging make Time-of-Flight SecondaryIon Mass Spectrometry (ToF-SIMS) a unique and powerful tool for biologicalanalysis. Many of these biological systems, including drug-cell interactions, requireboth the identification and location of specific chemicals. ToF-SIMS, used in imagingmode, is making great strides towards the goal of single cell and tissue analysis.The experiments, however, result in huge volumes of data. Here advanced computationalapproaches employing sophisticated techniques to convert these data intoknowledge are introduced.This thesis aims to produce a framework for data analysis, integrating novel algorithms,image analysis and 3D visualisation. New schema outlined in this thesisaddress the issues of the immense size of 3D image stacks and the complexity containedwithin the enormous wealth of information in ToF-SIMS data.To deal with the issues of size and complexity of ToF-SIMS data, new techniquesto processing image data are investigated. Automated compression routines for ToF-SIMSimages using a peak picking routine tailored for ToF-SIMS are evaluated. Newuser friendly GUIs capable of processing and visualising very large image stacks areintroduced as part of a tool-kit designed to streamline the process of multivariateanalysis and image processing. Along with this two well known classification routines,namely AdaBoost and SVMs, are also applied to ToF-SIMS data of severalbacterial strains to test their ability to classify SIMS data accurately. This thesispresent several new approaches to data processing and interpretation of ToF-SIMSdata.

Time-of-ight secondary ion mass spectrometry (ToF-SIMS) is a sensitive techniqueused to analyse the molecular composition of solid samples using keV ion beam sputtering.However only a small fraction (typically < 10^-3) of the desorbed material issecondary ions - the majority species (neutrals) cannot be extracted and detectedby the mass spectrometer. To increase the sensitivity and efficiency of the SIMStechnique, post-ionisation above the surface can be used. Lasers have been widelyemployed for molecular mass spectrometry due to the available high intensity, shortpulse width, high spectral purity and spatial coherence that allow them to be highlyfocused. For molecular samples the challenge is to achieve efficient post-ionisationwithout inducing extensive fragmentation, which limits the diagnostic value of theresulting mass spectrum. An investigation was performed into the ionisation anddissociation characteristics of a series of organic molecules under the action of intenselaser fields. This study is directed towards the analysis of biomolecules usinglaser post-ionisation. Here is reported progress towards the calibration of the experimentalset-up and mass spectral data from representative biomolecules in the gasphase. In this work a Ti:Sapphire laser was used with fundamental wavelength of800 nm and non-linear optical methods (OPAs) are used to extend the wavelengthinto the mid-IR region. System calibration is achieved using the ionisation of xenonatoms and comparing the results to established atomic tunnelling theory. This wasfollowed by the analysis of representative organic and biological molecules to studyionisation-dissociation characteristics. The molecules chosen were toluene, acetone,nitroaniline and histamine. A clear transition in behavior is observed favoringmolecular ion production. This behavior is discussed in the context of the underlyingmechanisms, and the implications for molecular post-ionisation analysis usingfocused ion beams.

Introduction: Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. Methods: In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. For this gas chromatography mass spectrometry (GC-MS) and ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) were used. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to elucidate novel metabolic pathways as potential therapeutic targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Results: Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers associated with hypoxia-induced chemoresistance were related to metabolic pathways including gluconeogenesis, DNA synthesis and fatty acid synthesis. Furthermore, network-based correlation analysis revealed specific metabolites in the fatty acid synthesis pathways were contributing to drug resistance, which included malonyl-CoA, 3-oxoeicosanoyl-CoA, stearoyl-CoA and octadecanoic acid. To facilitate the detection of metabolites in ToF SIMS datasets, a series of metabolites standard spectra were acquired. Hypoxic metabolite markers detected in ToF-SIMS data of cell lysates included glycine, lactic acid and succinic acid, which were also shown to be metabolite markers in GC-MS metabolic data. Furthermore, MTS sections were imaged using ToF-SIMS to profile the chemical response to chemotherapy treatment within the oxygen gradient. Loadings from image PCA were explored to determine the metabolic response in the highly oxygenated outer region and hypoxic inner region of the MTS. Conclusion: A multitude of analytical techniques were able to contribute to elucidating the metabolic mechanisms associated with hypoxia-induced chemoresistance. Metabolic profiling combined with a systems biology approach was further able to identify potential underlying metabolic regulation of resistance. Finally ToF-SIMS was developed as a tool for metabolite analysis in complex biological systems in situ. Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to identify novel metabolic pathway targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers of hypoxia-induced chemoresistance were discovered, which described the cellular metabolic response to chemotherapy resistance. Furthermore, network-based correlation analysis was used to construct metabolic networks to reveal novel metabolic pathways of the underlying metabolic response to chemotherapy resistance. Thus metabolic pathways were elucidated as potential therapeutic targets to overcome hypoxia-induced chemoresistance. In addition imaging mass spectrometry was developed as a tool to detect metabolites in situ. Metabolites were spatially localised within regions of a MTS to identify the metabolic response to hypoxia-induced chemoresistance in these 3D systems.

Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote.With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) in principle has the capability to detect and localise complex chemistry on the sub-cellular scale. Robust sample preparation protocols are therefore of great importance when attempting to preserve the natural biochemistry and architecture of biological specimens and extract meaningful information. It is likely that the optimum methodology will depend on the nature of the sample, and the goal of the analysis. The following study focussed on the preparation of cancer cells for ToF-SIMS analysis. Air drying, freeze fracturing, freeze drying and cytospinning-based preparation techniques and variations thereof were evaluated. Using image contrast, assessing spectra for the gain, or loss of native and non-native cellular species and the ratio of inorganic to organic ion yields, the investigation provides a systematic evaluation of the features of each preparation technique. This study then employed the optimum methods to determine the influence of the various phases of the cell cycle on classification of ToF-SIMS spectral data. A number of cell types, both cancerous and non-cancerous in G1, S and G2/M growth phases are separated by anticancer drug treatment and FACS (fluorescence activated cell sorting) and subjected to ToF-SIMS analysis. The results indicate that ToF-SIMS can detect surface biochemical changes induced by the cell cycle. Importantly it is also capable of detecting subtle alterations in cellular chemistry caused by anticancer drugs. Furthermore, the ToF-SIMS data was compared to results obtained from synchrotron sourced FTIR microspectroscopy to provide a more robust complementary analysis and support chemical evidence gathered.

Time-of-flight the secondary ion mass spectrometry (ToF-SIMS) is becoming a promising technique in analysis of a biological system due to its chemical specificity and the ability to perform high spatial resolution imaging. The combination of novel cluster and polyatomic beams has allowed generating images to map the distribution of biological components in tissue sections and cell surfaces. However, under these conditions the cluster beams have to be operated in the static regime, which limits sensitivity and confines the molecular imaging to 2 m spatial resolution. The polyatomic beams offer the benefits of high yields and low sub-surface damage, allowing the analysis to be performed at high ion fluence and abandoning the static limit. This presents a new approach to molecular imaging in which „voxels‟ are used rather than pixels, thus increasing sensitivity. As a result, the current SIMS instrumentation then becomes a limitation and has to be modified.A novel SIMS instrument, the J105 3D Chemical Imager has been developed with Ionoptika, which allows taking full advantage of the polyatomic primary beam, particularly the C60 by using it in a dc mode with buncher-ToF configuration. The aim of this project was to prove the concept and the potential of this new instrument.Initially standard organic samples have been used to show the current performance of the J105 for the analysis of organic samples with respect to a conventional ToF-SIMS instrument, the BioToF. The tandem MS capability of the instrument has been tested and proved using standard samples and a lipid mixture of brain extract.HeLa cells, an immortalised cell line were analysed using this instrument in 2D and 3D imaging mode. The images generated show molecular localisation on a sub-cellular scale, over a practical timeframe, whilst sustaining high mass resolution at 4000. The cells were imaged using a 40 keV C60+ dc beam and a clear differentiation between the material within the nuclei and lipid membrane can be made. Investigation of cell preparation suggested that the frozen-hydrated approach may be beneficial.The data presented in this thesis validates the new instrument concept offering the advantages of higher mass detection, increase in sensitivity, and an increase in the duty cycle while at the same time allowing imaging at sub-cellular scale with higher mass resolution.