Quantitative immuno-mass spectrometry imaging by laser ablation-inductively coupled plasma-mass spectrometry
- Publication Type:
- Thesis
- Issue Date:
- 2019
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Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an elemental bio-imaging technique that combines high sensitivity and spatial resolution of elements with quantification in two or three dimensions. LA-ICP-MS has recently been applied to detection and quantification of non-elemental analytes (eg. proteins) in tissue sections using immunohistochemical methods. This approach uses molecular probes such as antibodies, tagged with reporter elements such as the lanthanides which are not found naturally in biological samples. Analyses based on these methods is known as immuno-mass spectrometry imaging (iMSI) and has the potential for application to currently refractive research questions in cell and organ biology and new diagnostic platforms.
The drawbacks of commonly used matrix-matched tissue standards were addressed by development of novel facile methods for the preparation of moulded gelatin standards. Surface roughness and robustness were compared against cryo-sectioned gelatin and homogenised brain tissue standards. The moulded standards had significantly higher accuracy, precision and reproducibility and were easier to prepare. Additionally, background metals in gelatin were removed using chelating resins to increase the dynamic calibration range and to improve limits of analysis.
The resolution of LA-ICP-MS elemental bio-imaging is usually constrained by the diameter of the laser spot size and is often not adequate to explore in situ sub-cellular distributions of elements and proteins in biological tissue sections. Super-resolution reconstruction is a method used for many imaging modalities, combining multiple lower resolution images to create a higher resolution image. This thesis describes a super-resolution reconstruction method for LA-ICP-MS imaging by ablating consecutive layers of a biological specimen with offset orthogonal scans. Layer-by-layer image reconstruction was extended to the third dimension without the requirement of image registration across multiple sections. Quantitative super-resolution reconstruction provided superior image clarity and fidelity in two- and three-dimensions.
These methods were applied to the development of iMSI for both antibody and aptamer probes to quantify and localise dystrophin in muscle, and myelin basic protein in brain. Quantification of dystrophin is challenging by conventional methods and is central to development of Duchenne muscular dystrophy treatments. Samples were stained with a gadolinium labelled anti-dystrophin antibody and analysed by LA-ICP-MS. Normal mouse and normal human samples were found to have ~700 and ~300 parts per billion gadolinium respectively with under 20% relative standard deviation. The results improved on current methods and met FDA guidelines. The feasibility of using aptamers for iMSI was confirmed, which may enable analysis of challenging targets.
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