Characterisation of fluorescence nanotomography : a method for recovering the donor-acceptor distance distribution from FRET-modified fluorescence decays

• Main Author: Macnaught, Gillian
• Published: University of Strathclyde 2007
• Subjects: 535
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486535

Forster Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from an initially excited donor to an acceptor via a dipole-dipole interaction: FRET ~ modified donor fluorescence decays are dependent on the donor .:.. acceptor distance distribution function p(r) . This function contains valuable structural information regarding the slirroundings of the probe molecules. Currently the form of this function is assumed and then the calculated function is fitted to the measured decay via the Least Squares Method to determine the best fit parameters. Fluorescence Nanotomography (FN) is an ambitious technique developed within the Photophysics group that attempts ·to recover p(r) from FRET - modified fluorescence decays without making a speCific assumption regarding its form [1]. Instead p (r) is represented in tenns of a series of Laguerre polynomials. The ability to directly recover p (r) would make FN a po~erful structural sensing tool. However this· is a non - trivial technique requiring the solution of two inv'erse problems. 'The first is to. recover the pure' fluorescence response. ~ (1) from the measured fluorescence decay F (1). This problem was solved using the Maximum Entropy Method (MEM) which reconstructs the most probable lifetime distribution consistent with the measured decay. FN was then used to recover the form of p(r) from the MEM lifetime 'distribution. This thesis characterises' FN by applying it to calculated functions, synthetic decays and measured decays of Perylene and C02+ in glycerol and incorporated into silica gels, controlled Pore Glasses and in lipid bilayers. It was found that the physical information contained within the lifetime distributions recovered from the FRET modified decays using 'MEM is not adequate to enable the recovery of an absolute arb~trary donor - acceptor distance distribution. Therefore more specific prior information regarding the form of p(r) is required. .A more detailed study of the FRET kinetics ofPerylene and Co2 + encapsulated in a Tetramethyloxosilane (TMOS) gel is also presented here. This is analysed using the least squares method and illustrates the variety of factors affecting the FRET kinetics in a complex environment.