Lanterna Rally

Author: Michael Edward Kurczy

Publisher:

Published: 2009

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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.