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Fundamentals of Spectroscopic and Spectrometric Cellular Analysis
[Thesis]. Manchester, UK: The University of Manchester; 2011.
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Abstract
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) in principle has the capability to detect and localise complex chemistry on the sub-cellular scale. Robust sample preparation protocols are therefore of great importance when attempting to preserve the natural biochemistry and architecture of biological specimens and extract meaningful information. It is likely that the optimum methodology will depend on the nature of the sample, and the goal of the analysis. The following study focussed on the preparation of cancer cells for ToF-SIMS analysis. Air drying, freeze fracturing, freeze drying and cytospinning-based preparation techniques and variations thereof were evaluated. Using image contrast, assessing spectra for the gain, or loss of native and non-native cellular species and the ratio of inorganic to organic ion yields, the investigation provides a systematic evaluation of the features of each preparation technique. This study then employed the optimum methods to determine the influence of the various phases of the cell cycle on classification of ToF-SIMS spectral data. A number of cell types, both cancerous and non-cancerous in G1, S and G2/M growth phases are separated by anticancer drug treatment and FACS (fluorescence activated cell sorting) and subjected to ToF-SIMS analysis. The results indicate that ToF-SIMS can detect surface biochemical changes induced by the cell cycle. Importantly it is also capable of detecting subtle alterations in cellular chemistry caused by anticancer drugs. Furthermore, the ToF-SIMS data was compared to results obtained from synchrotron sourced FTIR microspectroscopy to provide a more robust complementary analysis and support chemical evidence gathered.