Shedding light on living cells and mineralised tissues using Raman spectroscopy
Creators: name: Cloyd, Kristy Lee
Publisher: name: Imperial College London
Year: 2013

description: Raman micro-spectroscopy presents a highly sensitive, non-invasive, and rapid way to collect biochemical information from cells and tissues. The resulting Raman spectrum is a chemical ‘fingerprint’ containing a wealth of molecular level information which has been used to characterize, monitor, compare and confirm biological processes from the cellular to tissue levels. The work presented in this thesis utilizes Raman spectroscopy to test live in vitro cellular models, classify human tissues of interest, and determine the biomolecular differences in tissue samples which are diseased or undergoing therapeutic treatment. Additionally new ways of visualizing and interpreting multivariate analytical results are proposed and demonstrated to ease the determination of the biomolecular features which are most important when comparing sample groups. A persistent challenge in the interpretation of information rich biological Raman spectra includes the multitude of signals from lipids, proteins, carbohydrates, nucleic acids, and minerals found in a limited spectral range and in some instances overlapping significantly. Partial Least Squares – Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores were presented as heat maps overlaying difference spectra to ease the visualization of significant biochemical bond changes between sample groups and their trends. The advantages of applying PLS-DA VIP scores in this way are demonstrated in well studied and known system including a cultured cellular model incorporating fixation methods and a human tissue comparison between healthy and osteoporotic bone. PLS-DA VIP score plots were additionally utilized to characterize and compare the biolomecular environments surrounding the recently described microscopic mineral inclusions in human aortic valves and aortae. The PLS-DA VIP score plots exposed the chemical differences in these systems through highlighting the corresponding spectral bands in an easy to read and interpret way. Raman micro-spectroscopy was also applied to investigate an in vitro ‘calcified’ porcine aortic valvular interstitial cell model. This model system was probed for the first time using the combination of Raman micro-spectroscopy and complimentary gold standard biological techniques to determine the protein and potential mineral content within these nodular, cellular systems. The ‘calcified’ porcine aortic valvular cell nodules showed no evidence of mineral inclusion. These nodules did exhibit a heavy extracellular matrix production including the production of collagen I. The porcine aortic valvular cell nodules acting as a model system for diseased aortic valve tissue requires not only the characterization of the cell nodule in vitro but also the characterization of the human disease spectrum which the model is suggested to replicate. The discovery and characterisation of microscopic mineral spherical inclusions (50nm-200µm) located in both valvular and vascular tissues leads to an interesting question on the introduction and role of microscopic mineral deposits in cardiovascular disease. Here Raman micro-spectroscopy was utilized to investigate the organic matrix surrounding these microscopic mineral deposits to determine if any colocalised protein changes exist. Protein and specifically collagen changes are demonstrated between tissues with and without the spherical mineral deposits despite being macroscopically indistinguishable. Raman spectroscopy was also utilized to provide direct insights into tissue constituent and structural changes on the molecular level in heat-induced tissue fusion via radio-frequency (RF) energy. This type of tissue fusion has gained wide acceptance clinically and is presented here as the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study exposed spectroscopic evidence for the loss of distinct collagen fibres rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. Raman spectroscopy is a demonstrated, powerful, biomolecular imaging technique which benefits from advancements in mathematical analytical techniques as well as its own application in biological investigations. This thesis explores the application of Raman spectroscopy in combination with powerful analytical techniques to further characterize and compare biological systems of interest.