Label free imaging of biological samples using mass spectrometry imaging and Stimulated Raman scattering microscopy

Majer, Jan 2023. Label free imaging of biological samples using mass spectrometry imaging and Stimulated Raman scattering microscopy. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Unlike fluorescence microscopy techniques, label-free imaging techniques avoid perturbation of the biological environment, which enable the analysis of samples in their native state. Label-free imaging methods for tissues and cells rely on the intrinsic properties of a sample to generate an image contrast. Laser desorption ionisation mass spectrometry imaging (LDI MSI) is a label-free technique that allows visualisation of sample composition based on analyte mass. Stimulated Raman scattering (SRS) microscopy, also label free, uses the vibrational properties of molecular bonds in a sample to generate images. This work describes the combination of LDI MSI and SRS imaging to improve the precision of the characterisation of the chemical components in a single sample based on their mass and vibrational properties. However, the conductive LDI slides required necessary for the ionisation process in LDI MSI, caused interference during SRS microscopy. The LDI-slide intrinsic interference required a replacement of LDI slides with standard non-conductive glass slides and an adhesive and conductive DIUTHAME membranes. Use of DIUTHAME bypassed the incompatibility issues and allowed the first described SRS and LDI MSI analyses of the same tissue sample. LDI MSI and SRS microscopy were successfully applied in the imaging of 2D cell cultures and complex in vitro models (CIVMs). SRS microscopy of heterocultures classified the individual cell types and cell phenotypes based on hyperspectral data from a small region of the Raman spectrum. The variability in the cell phenotype in hepatocyte CIVMs was also associated with differences in the intracellular antisense oligo (ASO) distribution. Thus, a successful approach for SRS-to-LDI MSI analysis of a single sample was achieved. This was applied in tissues and has great potential for applications in cell cultures and CIVMs. It is also demonstrated that SRS microscopy shows potential for the classification of cells based on their chemical composition

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Funders:	Horizon 2020
Date of First Compliant Deposit:	23 May 2024
Last Modified:	23 May 2024 13:27
URI:	https://orca.cardiff.ac.uk/id/eprint/169116

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