Development and Evaluation of Preparation Methods for the Analysis of Cell Membranes Using Time-of-Flight Secondary Ion Mass Spectrometry

Sammanfattning: The aim of this work was to evaluate and develop methods for preparing dry samples for TOF-SIMS analysis of cell membranes. For correct interpretation of the analysis data, an understanding of the effects that the preparation method has on the morphology and chemical structure of the sample must first be obtained. To achieve this, TOF-SIMS analyses, complemented by SEM and interference reflection microscopy (IRM), were performed on both real cells and artificial cell membranes in the form of supported phospholipid bilayers. Using supported POPC bilayers, a preparation method based on rapid cooling and subsequent freeze drying was developed and demonstrated to provide dry lipid bilayers for TOF-SIMS analysis. Furthermore, comparing different POPC layer structures, molecular and dimer ion yields of POPC were found to be 5-10 times higher for bilayers than for monolayers or disordered layers. This finding may be used to indicate the presence of intact lipid bilayers in artificial and real cell membranes. Different fixation and drying methods were applied to cells adhered to a silicon surface, and a method was developed in which the samples were washed in a solution of ammonium formate (AF), cryofixed by plunge freezing, and then freeze dried. The AF-wash/cryofixation method was successful in removing salt ions from the cell buffer yet preserving the potassium gradient at the cell membrane, and preserved cell membrane structure to a large extent, including the lipid bilayer structure. Chemical fixation by glutaraldehyde preserved fine cell membrane structures, below 100 nm, but at the same time negatively affected lipid bilayer structure and membrane integrity. Drying methods based on alcohol substitution removed membrane lipids, reducing the yield of phosphocholine by ~70 times. Fixation with osmium tetroxide was effective in retaining part of the membrane phospholipids. These results provide guidance on appropriate fixation and drying methods to be selected based on the analytical information requested.