Optimisation of a Real Time All Fibre Spectroscopic Fluorescence Probe

Abstract

Bacteria can be used to aid bioremediation and it is crucial to monitor the bacteria involved to gain feedback on the effectiveness of the remediation process. To allow monitoring, these bacteria can be labelled with fluorescent proteins. The optrode setup is an all fibre real time spectroscopic setup which allows in situ measurements of the bacteria fluorescence emission spectrum. The measured spectra can then be used to quantify the amount of bacteria present. The aim of this thesis was to make improvements on the optrode setup, first by understanding how different fibre parameters such as the numerical aperture and core diameter affect the illumination volume of the fibre probe. Photon propagation is modelled by Monte Carlo simulations, allowing the laser illumination volume to be visualised and the intensity decrease of the beam profiles to be plotted. The choice of fibre to make the probes can be decided depending on specific applications. To increase the detection limit of the setup, variations in spectral shape and amplitude of the acquired spectra need to be minimised. Existing experiment protocols were examined to isolate how variations in spectrum are introduced, and also how they can be avoided. Before the bacteria population can be quantified, the acquired spectrum needs to be separated into individual spectral components when several bacteria are measured together. Existing deconvolution algorithms were reviewed and a new method utilising the open-source software Fityk and the Levenberg-Marquardt optimisation was tested as an attempt to make the deconvolution process more versatile and applicable to a wider range of spectral shapes. Results of the optrode setup monitoring bacteria distribution and concentration are also presented to demonstrate the how the optrode setup can be used for different biomedical applications. In this research, the more intricate details of the optrode setup were investigated. The items presented in this thesis provide additional information regarding potential improvements of the optrode setup.