Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.3 PHYSICAL SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Technological University Dublin 2003.

Abstract

This research thesis describes the design, construction and optical testing of three fibre-optic microspectrophotometer systems to monitor the reduction of mitochondrial respiratory pigments. The optical and biochemical characteristics of mitochondrial respiration are discussed and the current optical techniques employed in the biochemical analysis of respiratory enzymes are presented. The primary focus of this study is the system parameters surrounding the spectrophotometric determination of mitochondrial respiratory activity. Three separate fibre-optic set-ups are described, which use visible spectrophotometry to measure the reduction of mitochondrial electron carriers from a range of biological sources. Each system is based around the Ocean Optics S1000/S2000 series microspectrometer. Factors, which wee found to affect the quality of the data produced, such as sample size, spectrometer specifications, data acquisitions, front end optical design and sample control (temperature and volume) were investigated. The primary aim was to develop a simple optical probe, which addresses the problem of the limited availability of the biological reference material. The key features of the systems are fast multispectral acquisition, high spectral resolution, font end adaptability and mechanical stability in micro samples. Data, acquired with each of the three separate systems, are presented for the specrophotometric characterisation of the respiratory pigment cytochrome-c using chemical reagents and in isolated mitochondrial samples, which represents a progression towards an increasingly in vivo biochemical environment with reduced sample volumes. Data is also shown for enzyme kinetics in permeabilised cultured cells for control cells and cells which have been chemically treated with ANAs to induce dysfunctional mitochondrial properties. Finally, date is presented for human muscle electron

DOI

https://doi.org/10.21427/D7GP5B

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