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.

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.

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.

The capability to characterize disease states by way of determining novel biomarkers has led to a high demand of single cell and organelle analytical methodologies due to the unexpected heterogeneity present in cells of the same type. Lipids are of particular interest in the search for biomarkers due to their active roles in cellular metabolism and energy storage. Analyzing localized lipid chemistry from individual cells and organelles is challenging however, due to low analyte volume, limited discriminate instrumentation, and common requirements of separation procedures and expenditure of cell sample. Using nanomanipulation in combination with mass spectrometry, individual cells and organelles can be extracted from tissues and cultures in vitro to determine if heterogeneity at the cellular level is present. The discriminate extraction of a single cell or organelle allows the remainder of cell culture or tissue to remain intact, while the high sensitivity and chemical specificity of mass spectrometry provides structural information for limited volumes without the need for chromatographic separation. Mass analysis of lipids extracted from individual cells can be carried out in multiple mass spectrometry platforms through direct-inject mass spectrometry using nanoelectrospray-ionization and through matrix-assisted laser/desorption ionization.