This thesis project consisted of two separate projects which were connected by roots in biological analysis. The first was to characterize the release kinetics of a protein from a biodegradable polymer membrane which had not previously been formulated on a scale large enough for clinical medicine. This scaled up membrane was assessed using Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and fluorescence spectroscopy for physical properties such as composition and release kinetics but was also analyzed using optical microscopy methods for biocompatibility. The second was to use of ToF-SIMS chemical imaging to determine the distribution of a drug called methylselenocysteine (MSC) in xenograft tumor tissue. MSC had previously been found to increase tumor vasculature which will in turn increase the distribution of CPT-11 throughout the tumor. This type of study had not previously been attempted with the ToF-SIMS chemical imaging method.^Based on the work done on both of these projects, three chapters are included in this thesis document and are summarized below. A new, low mass aggregation pattern was observed using ToF-SIMS for solid Aerosol-OT (AOT) thin films cast from concentrations above the critical micelle concentration (CMC) (10-3 - 10-5 M in chloroform). Conventional methods typically used to calculate the aggregation number of AOT include optical methods such as visible spectroscopy, dynamic light scattering, or infrared spectroscopy which use the appearance (or disappearance) of a pattern to discern the CMC. These methods all use a secondary molecule such as water to determine when a physical change in the AOT has occurred.^ToF-SIMS analysis allows for the direct analysis of an ion fragmentation pattern specific to the solution concentration of AOT to determine the CMC. Two molecularly distinct polymer membranes based on optimization of KGF activity were formulated to release biologically active KGF into solution. The protein that was used for this study is Keratinocyte Growth Factor (KGF). The membrane was formulated with an anionic surfactant AOT, which was hypothesized to contain the protein in an aqueous environment thereby preserving the activity during the formulation and delivery process. Each of these membranes was found to release a biologically relevant concentration of KGF and was able to sustain cell growth/adherence in vitro. ToF-SIMS chemical imaging was conducted using tissue that had been treated first with methylselenocystine (MSC) followed by the anticancer drug CPT-11.^ToF-SIMS analysis found that tumor and liver tissue that had been treated with MSC had characteristic ion signals that could be associated with this drug. Imaging with ToF-SIMS was confirmed with fluorescence microscopy and found that the drug segregated to the vasculature of the liver and tumor tissue.

Time of flight secondary ion mass spectrometric (ToF-SIMS) imaging is a powerful bioanalytical tool with the ability to produce molecular images of samples with submicron spatial resolution without the use of labels. In this thesis I will present the development of ToF-SIMS imaging methodology for biological analyses as well as applications that have yielded information about the role of lipids in membrane organization. In the first chapter, I introduce the plasma membrane and describe its fundamental role in maintaining life through the dynamic remodeling of its structure. I focus on two concepts that are believed to influence the localized chemical make up and structure of the membrane, intrinsic curvature and lipid domains. ToF-SIMS imaging is briefly described and a discussion of cluster ion bombardment and sample preparation is included. The chapter concludes with a survey of several important biological studies that have come out of the SIMS community. In Chapter 2 I report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative ToF-SIMS imaging was used to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In-situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single-cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases. Chapter 3 investigates prospects for three-dimensional SIMS analysis of biological materials using model multilayer structures and single cells. The samples were analyzed in a ToF-SIMS spectrometer equipped with a 20 and a 40 keV buckminsterfullerene (C60+) ion source. Specifically, molecular depth profile studies involving dehydrated dipalmitoylphosphatidylcholine (DPPC) organic films indicate that cell membrane lipid materials do not experience significant chemical damage when bombarded with C60+ ion fluences greater than 1015 ions/cm2. Moreover, depth profile analyses of DPPC?sucrose frozen multilayer structures suggest that biomolecule information can be uncovered after the C60+ sputter removal of a 20 nm overlayer with no appreciable loss of underlying molecular signal. The resulting depth information was used to characterize C60+ bombardment of biological materials. This information was used to controllably remove the plasma membrane of a single macrophage cell using a molecular depth profile approach allowing the analysis of the chemistry of the cytoplasm. Two methods that were developed to increase the reproducibility of biological SIMS analysis are covered in Chapter 4. First I demonstrate the utility of the C60+ cluster ion projectile for sputter cleaning biological surfaces to reveal obscured spatio-chemical information. 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. In fact, 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 did not alter the spatial distribution of the surface chemistry. This approach removes artifacts that are common to many biological sample preparation schemes for ToF-SIMS imaging. Removing these artifacts, which may obscure the surface chemistry of the sample, will increase the number of analyzable samples for SIMS imaging. The second method covered in Chapter 4 is freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment. This method was used to cryogenically preserve single cells for ToF-SIMS imaging analysis. 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 top most surface layers. In addition, I found water could be controllably re-deposited onto the sample at temperatures below -100 oC 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 we have obtained high quality images and spectra with both atomic bombardment as well as C60+ cluster ion bombardment. Sample handling is also the topic of Chapter 5. It this chapter, I describe a device which has been designed to prepare frozen, hydrated single cell cultures with a freeze fracture methodology for ToF-SIMS analysis in an ION-TOF (GmbH) TOF-SIMS IV mass spectrometer. The device reproducibly produces frozen hydrated sample surfaces for SIMS analysis. I show that SIMS analysis with the Bi32+ produces high-resolution molecular images of single PC12 cells in an ice matrix. I also show that the combination of ionization enhancements that are provided by both the ice matrix and the cluster ion source facilitates the localization of lipid ions that have not been localized in these cells previously. Namely, two fragments of phosphatidlyethanolamine (m/z 124 and m/z 142) and a large fragment of phosphatidylcholine (m/z 224). The ability to localize and measure these ions will increase the number of question that SIMS imaging can be used to answer. In Chapter 6 ToF-SIMS imaging was used to demonstrate that lipid domain formation in mating single-cell organisms is driven by changes in membrane structure. Studies of lipid bilayers in both living and model systems have revealed that lipid composition is coupled to localized membrane structure. However, it is still not clear if the lipids that compose the membrane actively modify membrane structure or if it is structural changes that cause lipid heterogeneity. I report that time of flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired before, during and after mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, does not precede structural changes in the membrane. Rather, domains are formed in response to function during cell-to-cell conjugation. ToF-SIMS imaging has been used to collect information with wide implications in all membrane processes. The work presented here is the continuation of a project aimed at chemically characterizing biological samples with spatially resolved mass spectra, with a particular focus on single cell imaging. Much of the work I have done has centered on understanding the capability of current technology and using this understanding to solve a particular problem. This work is vital to keeping SIMS in the biological realm but the development of new technology is the ultimate future for these experiments by increasing the number of tools that the experimenter has to choose from. In Chapter 7 discuss two ongoing projects that I think will lead to the next break through bringing us closer to realizing the goal of this project: a complete chemical map of a single cell.

This book provides an overview of the phenomenology, technology and application of secondary ion mass spectrometry as a technique for materials analysis. This approach is developing into one of the most effective methods of characterizing the composition and chemical state of the surface and sub-surface layers of solid materials. The first three chapters introduce the basic physical and chemical principles involved and the theories which have been proposed to explain the process. Subsequent chapters describe the instrumental components of the SIMS apparatus, the use of SIMS as an analytical tool, and the development of the techniques of sputtered neutral mass spectrometry and laser microprobe and plasma desorption mass spectrometry. Many practical examples are featured to illustrate the application of SIMS to real problems, possible pitfalls are pointed out, and data of use to analysts are collected in appendices. The book is a practical guide suitable for scientists in all fields who wish to use this valuable analytical technique.

Following the biannual meetings in MUnster (1977) and Stanford (1979) the Third International Conference on Secondary Ion Mass Spectroscopy was held in Budapest from August 31 to September 5, 1981. The Conference was attended by about 250 participants. The success of the 1981 Conference in Budapest was especially due to the excellent preparation and organization by the Local Organizing Committee. We would also like to acknowledge the generous hospitality and cooperation of the Hungarian Academy of Sciences. Japan was chosen to be the location for the next conference in 1983. SIMS conferences are devoted to two main issues: improving the application of SIMS in different and especially new fields, and understanding the ion formation process. Needless to say, there is a very strong interaction be tween these two issues. The major reason for the rapid increase in SIMS activities in the last few years is the fact that SIMS is a powerful tool for bulk, thin-film, and surface analysis. Today it is extensively and successfully applied in such different fields as depth profiling and imaging of semiconductor devices, in isotope analysis of minerals, in imaging biological tissues, in the study of catalysts and catalytic reactions, in oxide-layer analysis on metals in drug detection, and in the analysis of body fluids.

Developing tools to elucidate the chemical distribution of lipid components within the eukaryotic cellular membrane is critical to understanding their role in many cell processes. Secondary ion mass spectrometry (SIMS) is a technique that offers both chemical and spatial specificity, and has become popularized over the last decade for analyzing model and native cellular membranes. Herein, this thesis describes the use and development of SIMS for such samples. By employing high-resolution SIMS, performed on a Cameca NanoSIMS 50, and atomic force microscopy (AFM) the influence of cholesterol on the phase behavior of supported lipid membranes containing saturated phosphatidylcholine lipid species was studied. While the NanoSIMS 50 afforded unprecedented lateral resolution on the chemical distribution of these model membranes, it was achieved at the cost of employing stable-isotope labels for component identification. Time-of-flight SIMS (TOF-SIMS), on the other hand, is a molecular imaging technique that does not require the use of labeled species. However, the ability to image characteristic lipid fragments (i.e. lipid headgroups, etc.) at lateral resolutions comparable to the NanoSIMS 50 is challenging. Furthermore, many of the characteristic fragments are common between structurally similar lipids, such as different phosphatidylcholine species, making discrimination between these species difficult. This challenge was overcome by developing a multivariate analysis (MVA) method, called principal component analysis (PCA), for evaluating the TOF-SIMS spectra of these samples. As a result, the ability to image and identify saturated phosphatidylcholine lipids that differ only in chain length within phase-separated membranes was achieved and could be registered to the corresponding AFM image. By performing PCA to compare TOF-SIMS spectra of labeled and unlabeled species, the molecular ion peaks that are associated with these phosphatidylcholine lipids were identified. These known ion peaks were then used to optimize PCA for TOF-SIMS imaging of phase-separated supported lipid membranes to attain a greater lateral characterization of these samples. The ability to gain quantitative information from TOF-SIMS analysis of homogenous supported lipid membranes was made possible by performing partial least squares regression (PLSR) on the resulting mass spectrum. Here, calibration samples were modeled, and then used to quantitatively predict the content of unknown membrane samples. Lastly, a TOF-SIMS MVA approach was utilized to evaluate native cellular membranes with the goals of differentiating between cell types, and in a separate project, identify the binding of vascular endothelial growth factors to human endothelial cells.