Examining biological systems at a molecular level via polarised light spectroscopy and scattering turbidity

During the course of this work the development and optimisation of the dichroism field of spectroscopic techniques regarding their application towards living biological systems has been examined. To this end we have studied the theory of light scattering, as one of the principle flaws that can prevent accurate analysis of spectral data. The fields of Rayleigh-Gans-Debye and Mie theory were investigated and implemented into a program for the calculation of scattering for various different systems. The developed program was then optimised using previously collected data regarding the scattering produced by liposomes under flow, and further data regarding the deformation of liposomes validated. A secondary feature to be addressed was the inherent complexity of cellular spectra that comprise a variety of different components. Experimental studies were therefore conducted regarding the deconvolution of several biological systems in terms of their individual chromophores, in increasing degrees of complexity. An approach in which each component was converted into a series of Gaussian curves and the contribution of sets of these Gaussians (from each component) to the overall system spectrum was developed. Since biological systems contain various components that interact with each other, such as the hypochromicity of DNA, the influence on this spectral decomposition approach was also outlined and studied. Unusual features were observed in the most complex cellular samples of red blood cells and fission yeast, regarding the linear dichroism (LD) spectra produced in the absence of flow. Further studies were conducted regarding the simpler of these systems, red blood cells, to determine the source of these spectral features. The principle component was determined to be oxy-hemoglobin, and that both cellular and protein orientation was required to produce the observed LD spectrum. Sedimentary forces were then discovered to be the signal source within the sample vessel, and the spectrum comprised of both absorbing and scattering elements. Of the latter, both typical RGD/Mie type scattering was observed in addition to resonance scattering. In addition, it was observed that alterations to the cellular geometry, due to various environmental influences such as pH and tonicity, were directly related to spectral behaviour