Towards chemical profiling at the cellular level

Traditional methods for the analysis of cellular components have focused on 'grid and find' assays that provide quantitative information from a large population of cells, often as many as a million cells. The results of these studies are often presented only as the percentage of the dry weight of the cells and not the concentration within individual cells. The research presented in this thesis is concerned with the development and application of methods for single cell sampling and analysis (SiCSA) from fungal cells that overcomes this deficit. The methods developed offer the potential to investigate the intra-cellular concentration of biologically relevant molecules within selected cells of a heterogeneous population. The instruments and techniques for this work are described along with an overview of the fundamental principles behind this methodology. The model organism studied in this work was the filamentous fungi, Neurospora crassa, the orange bread mould. It is the best characterised of all the filamentous fungi, a group of organisms that are critically important to agriculture, medicine and the environment. Capillary electrophoresis electrospray mass spectrometry (CE-ESIMS) was used to measure the intra-cellular concentration of disaccharides, in particular trehalose. In Neurospora crassa this molecule is synthesised in response to environmental stress, and has been reported to accumulate at concentrations as high as 10 mM, based on measurements using bulk cell populations. The value of 1.3 mM for the intra-cellular concentration of disaccharide measured in the single cell sampling experiments described in this thesis is in good agreement with this previously published maximum concentration. Following topical application of a commercially relevant fungicide, azoxystrobin, to cell cultures of Neurospora crassa, the intra-cellular concentration of the fungicide was measured. For cells treated with azoxystrobin at a concentration of 14.8 pM (the saturation concentration of azoxystrobin in water), the intra-cellular concentration was shown to reach 9.9 μM within 5 minutes. It is likely that the high surface to volume ratio of the fungal hyphae facilitats the rapid diffusion of these large hydrophobic molecules across the cell membrane. The development of novel instrumentation applicable to the analysis of ultra-low volume samples is presented, encompassing microsampling, transfer, ionisation and detection. Their utility in comparison with competing techniques is discussed, along with suggestions as to the expected development of this technique and possible directions for future work