Elemental bio-imaging : in situ analysis of trace elements in tissue by laser ablation-inductively coupled plasma-mass spectrometry

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Elemental bio-imaging is a new application of laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) that determines in situ trace element concentrations in thin sections of biological tissues. This project developed a LA-ICP-MS method for creating colour images of the regional distribution of both metals and non-metals in a variety of biological samples. The developed method was capable of producing images with a lateral resolution as low as 30 µm. This was achieved using a 30 µm laser spot that was rastered across the sample at a rate of 30 µm s⁻¹. It was found that a laser fluence of 0.22-0.28 J cm⁻² was best suited for soft tissue sections with minimal particle redeposition and fracturing of the surrounding sample. Evaluation of the octopole reaction system (ORS) fitted to the Agilent 7500 ce instrument found that the use of a collision gas for interference removal was inadvisable for imaging experiments. Experiments were carried out to determine the significance of potential polyatomic interferences in the ablation of tissue. It was found that the 'dry' nature of the plasma in LA-ICP-MS significantly reduced the occurrence of O and H based polyatomics, and the small sample load in each ablation reduced the effect of other matrix-based interferences on elements in the mg kg⁻¹ concentration range. Application of the method to sections of human lymph nodes impregnated with malignant melanoma found imaging of ³¹P was able to accurately discern healthy cells from cancerous tissue. Measurement of the ratio between ³¹P and other elements improved the contrast between the two types of cells. Three-dimensional models of imaged lymph nodes further improved the distinction between the melanoma cells and normal tissue. A method was developed for producing matrix-matched tissue standards by homogenising chicken tissue with known amounts of added elemental standards. Digestion of the tissue standards was performed and analysis by solution nebulisation ICP-MS confirmed the concentration of each added element. The standards were frozen and cut to the desired thickness for ablation and construction of multi-point calibration curves. An Fe-fed mouse model for Parkinson's disease (PD) was used to demonstrate the characteristics of the technique. C57BL6 mice were fed a diet high in Fe during development and were treated with both clioquinol (CQ) and L-DOPA, both of which are thought to chelate Fe in the brain. Results showed a decrease in Fe in the treated animals within the region of the brain called the substantia nigra (SN), which is adversely affected in PD. A 6-hydroxydopamine (6-OHDA) model for PD was also examined. 6-OHDA is directly injected into the rodent brain, stimulating the loss of cells within the SN. Imaging of sections taken from 6-OHDA lesioned animals showed a significant increase in Fe within the SN bilaterally when compared to control animals. In summary, elemental bio-imaging is a new method that can be applied to tissue sections from many sources, including humans. The technique has the potential to assist biologists in identifying possible new biomarkers for disease, related specifically to trace elements.
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