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.