AbstractPossessing high three dimensional optical sectioning capabilities and deriving chemical contrast from the intrinsic molecular vibrations of the sample, coherent anti-Stokes Raman scattering (CARS) microscopy has the ability to deliver high sensitivity non-invasive biological imaging. It is, however, accompanied by a deleterious non-resonant background (NRB) which acts to reduce the contrast and severely complicate analysis. Computational approaches are currently favoured for removing this NRB; however, these result in significant spectrally varying errors. This thesis concerns the development and subsequent implementation of a CARS platform employing a novel, all-optical, non-resonant background removal mechanism: Spectral Interferometric Polarisation Coherent Anti-Stokes Raman Scattering (SIPCARS). Exploiting the phase change that accompanies a Raman resonance and employing an elliptical pump/probe beam and linear Stokes beam, SIPCARS allows the complete removal of the NRB. The resulting SIPCARS spectra encode mode symmetry information into the amplitude response which can be directly related to polarisation resolved spontaneous Raman scattering spectra.
Verification of the SIPCARS methodology was achieved using spectra acquired from pure liquid samples which were in complete agreement with the corresponding polarisation resolved spontaneous Raman scattering spectra. The multiplexing limit of the system was assessed using several multi-component polymer bead mixtures and a lower limit of four determined. High signal-to-noise ratio SIPCARS imaging of a HeLa cell in the vibrational fingerprint region was acquired, from which it was possible to identify lipid droplets and subsequently, by producing ratio images, assess their degree of lipid unsaturation and the level of oxidised lipid content. The effect of a naturally derived phytotherapeutic lipid metabolism altering drug on the lipid droplets, contained within wild type N2 Caenorhabditis elegans nematodes, was addressed using SIPCARS. Assessing lipid unsaturation and area fraction, the drug was shown to produce a marked effect: a significant reduction in storage of saturated fatty acids post exposure. Additionally the ability of SIPCARS to differentiate between a variety of different C. elegans mutants was also demonstrated.
SIPCARS currently provides perhaps the only viable route to attain truly quantitative NRB-free CARS data; however, expanding on the foundation provided by this thesis, and following further development, it has the potential for profound implications in a wide range of areas including fundamental life sciences research, novel drug characterisation and histopathology.
|Date of Award
|David Richards (Supervisor), Bradley Littleton (Supervisor) & Frederic Festy (Supervisor)