Using single molecule spectroscopy to study fast photoprotective processes in plants

Abstract

The fundamental mechanisms involved in photosynthesis provide an opportunity to study physical principles that span over both classical and quantum scales. A better understanding of these mechanisms will benefit the development of alternative energy sources such as cheaper biofuel and more effective photovoltaics. This dissertation describes the single molecule spectroscopy setup that was assembled during my MSc-degree and the underlying theory required to understand the technique, is discussed. The greatest part of the setup development involved customised software development that performs the measurement. The code of this software is briefly discussed. Thereafter the results of a series of single molecule spectroscopy measurements of isolated light harvesting complex II (LHCII) that undergo non-photochemical quenching (NPQ) are described. The fast, reversible, energy-dependent component (qE) of NPQ is emulated by lowering the pH of the solvent in which the complexes are diluted. Apart from fluorescence intensity measurements, time correlated single photon counting is used to measure fluorescence lifetimes, which serves as an indirect measurement of NPQ. It was found that quenching could be taking place before the energy reaches the terminal emitter, and a relationship between intermediate fluorescence states and high jumping frequencies was established.Die fundamentele meganismes wat by fotosintese betrokke is skep 'n ideale geleentheid om beginsels te bestudeer wat oor beide klassieke en kwantumskale strek. 'n Beter verstaan van hierdie meganismes sal die ontwikkeling van alternatiewe energiebronne soos goedkoop biobrandstof en meer effektiewe fotovoltaïese selle bevorder. Hierdie verhandeling beskryf die enkelmolekuulspektroskopie-opstelling wat tydens my MSc-graad opgerig is en die onderliggende teorie wat nodig is om die tegniek te verstaan, word bespreek. Die grootste deel van die ontwikkeling van die opstelling het die ontwikkeling van toepassingsgerigte sagteware behels. Die kode van hierdie sagteware word oorsigtelik bepreek. Vervolgens word die resultate van 'n reeks enkelmolekuulspektroskopie-metings beskryf waartydens nie-fotochemiesedowing (NFD) in die geïsoleerde ligversamelingskompleks II (LHCII) van hoër plante bestudeer is. Die vinnige, omkeerbare, energie-afhanklike komponent (qE) van NFD is geëmuleer deur die pH van die oplossing waarin die komplekse opgelos is, te verlaag. Buiten metings van die fluoressensie-intensiteite is tydsgekorreleerde enkelfotontelling ook toegepas om fluoressensieleeftye te meet, wat as 'n indirekte meting van die mate van NFD dien. Die moontlikheid dat dowing plaasvind voordat die opwekkingsenergie die laagste energietoestand in die kompleks bereik, is ontdek en 'n verwantskap tussen intermediêre fluoressensietoestande en hoëfrekwensieskakeling word gelê.