Advancement of laser-based atomic spectroscopy techniques for biological sample analysis; from elemental profiling of tissues down to the cell organelle level
Creators: name: Davison, Claire Louise
Publisher: name: University of Surrey
Year: 2025
Subjects: Single cell analysis
Atomic Spectroscopy

description: Metals play a key role in fundamental structural and metabolic processes at the tissue, cell and organelle level. Consequently, elevated and unregulated concentrations of certain metals have been linked to the presence and progression of disease. Metal dysregulation can cause adverse pathogenic effects, but deliberate metal imbalances can also be used by the host as a response mechanism to infection. Studying molecular and elemental differences between healthy and non-healthy biological tissue can improve our mechanistic understanding of pathogenesis and consequently, novel approaches to disease treatment can be developed. Furthermore, by identifying sites of metallodrug accumulation and using this information to optimise dose, undesirable side effects can be reduced and therapeutic outcomes improved. The development of highly specialised analytical tools is therefore crucial for answering increasingly complex biological questions at the tissue, cellular and subcellular level. The work described here had the objective of expanding the boundaries of laser based atomic spectroscopy techniques, specifically single cell–inductively coupled plasma–mass spectrometry (SC-ICP-MS), laser ablation-ICP-MS (LA-ICP-MS) and laser induced breakdown spectroscopy (LIBS), for biological sample analysis. Emphasis was placed on the importance of sample preparation and introduction approaches to broaden the range of biological applications of each technique. The research was divided into three sections of increasing analytical complexity. First, a method for the high throughput elemental analysis of mammalian monocytes and macrophages infected with a containment level 2 model bacterium using SC-ICP-MS was developed. Significant elemental heterogeneity (Mg, Zn, Ca) in a macrophage model of tuberculosis was identified and this work addressed the analytical barriers, specifically with regards to sample preparation, that have previously restricted the advancement of the technique into the research branch of bacterial infections. Chemical fixation was identified as being crucial in the analysis of mammalian cells prior to sample introduction so that they can be resuspended in a solution suitable for SC-ICP-MS. It was shown that different fixatives (methanol 60–100% in H&lt;sub&gt;2&lt;/sub&gt;O and 4% paraformaldehyde in phosphate-buffered saline solution) impact measured cell elemental composition (Mg, Zn, Ca, Mn). Secondly, an additional layer of instrumental complexity in the form of spatial elemental mapping was incorporated into the project. A systematic optimisation was carried out to determine the practical feasibility of combining LA-ICP-MS and LIBS for the spatial mapping of endogenous elements in biological tissue, with emphasis placed on achieving improved accuracy of spatial maps by use of tandem normalisation techniques. Finally, cellular and subcellular localisation of analytes was achieved through the coupling of LA with nanocapillary sampling, applied to study uptake of radiopharmaceuticals. This research spans the fields of applied analytical chemistry, bacterial infections and cellular pharmacokinetics, with significant advances made in the development of analytical tools which can answer increasingly complex biological questions.