In this work the chemical analysis of biological cells and tissues with time-of-flight secondary ion mass spectrometry (ToF-SIMS) was explored. ToF-SIMS has the ability to obtain a mass spectrum with submicron spatial resolution for imaging and is extremely surface sensitive. ToF-SIMS for biological sample analysis is still an emerging field, so the development and characterization of novel sample preparation and analysis methods is key to acquiring useable information. In this work, three different methods to prepare NIH/3T3 fibroblasts were investigated: chemically fixed, freeze-dried and frozen-hydrated. Chemical fixation followed by rinsing removed a majority of intracellular Cl-, improving the secondary ion yields of all organic positively charged secondary ions an average of 2.6x. Damage cross sections were reduced during frozen-hydrated analysis, improving the secondary ion yields of higher mass organic fragments. In a separate experiment, accurate 3D reconstructions of NIH/3T3 fibroblasts were produced. A simple z-correction was applied to the data cube, and the biggest assumption for that correction was validated. An intracellular lipid-rich region surrounding the nucleus was visualized. ToF-SIMS applied to two different breast cancer systems. In the first, eight human breast cancer cell lines were distinguished from one another using mass spectra and principal component analysis (PCA). Not only was PCA to distinguish the cell lines form one another, it also highlighted the largest sources of variance between the cells. Phosphocholine, fatty acids, cholesterol and diacylglycerols (DAGs) were identified as key peaks. The identification of these species indicate that differences in lipid metabolism play an important role in separating the cell types from one another. Breast cancer tumor tissues were also investigated. Data from four tumors was collected. PCA applied to the spectra distinguished the four tissues from one another. Imaging PCA determined the largest sources of variance within an analysis area. Structures were identified by PCA that matched structures observed in serial-sectioned, conventionally-stained slices, and other domains that were not visible in the conventionally-stained slices. As with the breast cancer cell lines, phosphocholine, fatty acids, DAGs, cholesterol and vitamin were found to be large sources of variance, indicating lipid metabolism plays in important role in tumor differentiation.

This book highlights the application of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) for high-resolution surface analysis and characterization of materials. While providing a brief overview of the principles of SIMS, it also provides examples of how dual-beam ToF-SIMS is used to investigate a range of materials systems and properties. Over the years, SIMS instrumentation has dramatically changed since the earliest secondary ion mass spectrometers were first developed. Instruments were once dedicated to either the depth profiling of materials using high-ion-beam currents to analyse near surface to bulk regions of materials (dynamic SIMS), or time-of-flight instruments that produced complex mass spectra of the very outer-most surface of samples, using very low-beam currents (static SIMS). Now, with the development of dual-beam instruments these two very distinct fields now overlap.

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) can be utilized to map the spatial distribution of small molecules on a surface with potentially submicron resolution. Due to the inherent characteristics of this technique and its potential to provide higher spatial resolution than light microscopy based techniques without the use of chemical labels, it has been utilized to study the distribution of phospholipid species in the cell membrane. It is now known that many cell membranes contain transient compositional heterogeneities, colloquially referred to as domains, which participate in vital physiological processes such as exocytosis and signal transduction. Because of their size and lifetime, much remains unknown about the nature of these heterogeneities. ToF-SIMS imaging combined with cryogenic sample preparation techniques is a promising analytical platform poised to contribute greatly to this growing field of study. Sample preparation is crucial to obtaining quality lipid distribution maps, especially when dealing with single biological cells. To achieve this end the Winograd and Ewing groups have developed a freeze-fracture methodology adapted from cryo-SEM studies. Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has also been extensively used in conjunction with electron microscopy. This technique has been applied to ToF-SIMS imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. I demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, I found that water can be controllably re-deposited onto the sample at temperatures below -100° C in vacuum. The re-deposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, high quality spectra with both atomic bombardment as well as C60+ cluster ion bombardment, have been obtained. To date, many cell imaging studies have concentrated on phosphatidylcholine distributions, owing to its abundance and high ionization efficiency. However, cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various bio-molecules. Due to the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. MS/MS data obtained from a prototype C60+/ quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional ToF-SIMS spectrum. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging sub-cellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With ToF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows for pixel-to-pixel intensity comparisons and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated here by imaging the cell membrane of resting RBL-2H3 mast cells. Mass spectrometry imaging has been used to demonstrate that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. ToF-SIMS images of mating Tetrahymena thermophila, acquired at various stages during mating, can be used to demonstrate that lipid domain formation follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes. There is considerable interest in the unique properties of cluster ion projectiles and investigations of how they may be utilized to improve biological imaging. A C60+ cluster ion projectile was employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning; a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C60+ bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that may obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging. In general, out results show that by removing these artifacts, the number of analyzable samples for SIMS imaging is greatly expanded. Although imaging with sub-cellular spatial resolution has been demonstrated, it is clear that the success of future experiments is limited by the ionization efficiency of the lipids, as well as limitations imposed by a coaxial ToF geometry. Considerable work has been done in the lab, to address these limitations. This effort has resulted in the development of a hybrid quadrupole orthogonal ToF instrument equipped with a C60+ primary ion source. The capabilities and potential of this new platform will greatly increase the contributions of SIMS to the biological sciences.

Food contains various compounds and many technologies exist to analyze those molecules of interest. However, the analysis of the spatial distribution of those compounds using conventional technology, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry is difficult. Mass spectrometry imaging (MSI) is a mass spectrometry technique to visualize the spatial distribution of molecules, as biomarkers, metabolites, peptides or proteins by their molecular masses. Despite the fact that MSI has been generally considered a qualitative method, the signal generated by this technique is proportional to the relative abundance of the analyte and so quantification is possible. Mass Spectrometry Imaging in Food Analysis, a volume in the Food Analysis and Properties Series, explains how the novel use of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) will be an ideal complementary approach. MALDI-MSI is a two-dimensional MALDI-MS technology that can detect compounds in a tissue section without extraction, purification, separation, or labeling. It can be used to visualize the spatial distribution of biomolecules in foods. Features: Explains the novel use of matrix-assisted laser desorption/ionization mass spectrometry imaging in food analysis Describes how MALDI-MSI will be a useful technique for optical quality assurance. Shows how MALDI-MSI detects food contaminants and residues Covers the historical development of the technology While there are a multitude of books on mass spectrometry, none focus on food applications and thus this book is ideally suited to food scientists, food industry personnel engaged in product development, research institutions, and universities active in food analysis or chemical analysis. Also available in the Food Analysis and Properties Series: Food Aroma Evolution: During Food Processing, Cooking, and Aging, edited by Matteo Bordiga and Leo M.L. Nollet (ISBN: 9781138338241) Ambient Mass Spectroscopy Techniques in Food and the Environment, edited by Leo M.L. Nollet and Basil K. Munjanja (ISBN: 9781138505568) Hyperspectral Imaging Analysis and Applications for Food Quality, edited by N.C. Basantia, Leo M.L. Nollet, and Mohammed Kamruzzaman (ISBN: 9781138630796) For a complete list of books in this series, please visit our website at: www.crcpress.com/Food-Analysis--Properties/book-series/CRCFOODANPRO

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is the most versatile of the surface analysis techniques that have been developed during the last 30 years. This is the Second Edition of the first book ToF-SIMS: Surface analysis by Mass Spectrometry to be dedicated to the subject and the treatment is comprehensive

Time of flight secondary ion mass spectrometry, TOF-SIMS, is a highly surface sensitive analytical technique that can provide information about composition with submicron lateral resolution for a wide variety of materials. In conjunction with the latest cluster ion sources, organic depth profiling is also commonly performed now. For select materials, TOF-SIMS provides unparalleled sensitivity along with excellent reproducibility, and as a mass spectrometric technique, it also provides excellent specificity in the identification of many organic materials. Of the analytical methods available, it is among the most surface sensitive, but the physical principles that underlie it are also the least understood. This volume describes the instrumentation, the physical principles behind the technique to the extent they are understood, and provides a practical approach for the interpretation of TOF-SIMS data. The use of advanced data processing methods such as multivariate statistics are described in a readily approachable manner along with guidelines to help the reader understand where they are or are not really helpful. Given a basic background in undergraduate chemistry and physics, the book will be of use to any student with an interest in the technique. While the analyses are in fact performed in a vacuum, they are conducted in the context of a wider laboratory environment where many other analytical methods are available. The place of TOF-SIMS amongst them, when it is appropriate to use this method or another, or when multiple methods should be used in conjunction with TOF-SIMS is discussed in some depth. Examples of the wide range of applications of TOF-SIMS for research and problem solving in Academic Laboratories, National Laboratories, and Industrial laboratories, as it is applied to polymeric, biological, semiconductor, metallic, insulating, homogeneous, and inhomogeneous surfaces are described.