Development and characterization of fibre-based systems for intramural functional fluorescence imaging

Abstract

This thesis deals with three separate studies relating to the development and refinement of novel fibre optic-based techniques for an intramural fluorescence recording of electrical activity in the heart. Cardiac fluorescence measurements are affected by motion of the heart wall with respect to the imaging system. Electro-mechanical uncouplers, such as 2-3 butanedione monoxime, are widely used to reduce motion artifact in cardiac optical mapping, but these agents also depress the electrical function of the heart. A dual wavelength subtraction method has been developed that enables action potentials to be extracted from dual wavelength signals acquired with ratiometric voltage-sensitive dyes. This approach has been validated using fluorescence imaging recorded with an intramural fibre optic probe in an isolated supported pig heart preparation. It provides a means of reducing the concentration of uncoupler used for optical recording of cardiac electrical activity. A systematic study of illumination and effective fluorescence collection fields for fibre optic probes in various media, including tissue, has been completed using novel techniques. These include two-photon scanning confocal microscopy and two-photon flash photolysis with caged fluorophores. A semi-empirical model which describes the illumination and fluorescence collection profiles of multi-mode optical fibres was also developed. It has been shown that the effective fluorescence collection volume of fibre optic probes is predominantly determined by fibre diameter and the optical properties (mostly scattering) of the medium. Further investigations of the factors that give rise to delay in the upstroke of action potentials recorded using optical techniques suggest that this is not simply due to the spread of activation across the collection volume addressed by the imaging system as has previously been argued. Finally a prototype fluorescence imaging system incorporating modular fibre optic components and solid state lasers has been developed and characterized. The results obtained demonstrate the advantages of this approach in comparison to conventional systems that use free-space optics. The modular system is robust and compact, and optical alignment is not required to avoid cross-talk between channels. The prototype multi-channel system was successfully tested in an experimental setting using a 532 nm solid state laser. The approach outlined in this thesis provides a basis for future construction of a multi-optrode imaging system that is both relatively simple and feasible in terms of the costs involved.