The application and analysis of single-molecule spectroscopy data in photosynthesis

Abstract

Most of the energy that sustains life on earth can be traced back to photosynthesis, and so its importance is difficult to overstate. Our understanding of this crucial process is still growing as we develop ever-advanced techniques to investigate the world of proteins and their interactions with light. Single-molecule spectroscopy (SMS) has emerged as a transformative technique for studying molecular systems at the nanometre scale, offering unparalleled insights into dynamic processes and heterogeneity at the individual molecule level. However, effective utilisation of SMS data often requires sophisticated analytical tools and stable experimental setups, which can pose significant challenges to researchers. This thesis addresses these challenges through the development of Full SMS, a versatile analysis software suite and a custom-designed sample cell holder, which together provide an integrated framework to advance SMS-based studies.

Full SMS is a comprehensive graphical user interface (GUI)-driven software package for processing and analysing multiparameter SMS datasets. These datasets typically include fluorescence intensity and lifetime, as well as spectral properties of single dye molecules, quantum dots, or fluorescent active biomolecules. The software enables detailed analysis through tools for statistical evaluation of fluorescence intensity, clustering, and level identification; lifetime decay fitting; and second-order correlation function calculations. Visualisation features include the display of fluorescence intensity and spectral traces, as well as raster-scan images, with robust filtering options to tailor data processing. A custom HDF5-based file structure ensures efficient storage, while flexible export capabilities facilitate integration into broader research work flows. Written in Python, Full SMS is open source and accessible to users without programming expertise, leveraging a multiprocess architecture for enhanced computational performance. Full SMS is used to analyse three sample types as case studies to showcase its utility and ease-of-use. Complementing the software, this thesis also presents a custom-designed sample cell holder, which extends the capabilities of the SMS experimental setup designed and assembled in-house at the University of Pretoria. This hardware innovation enhances stability and versatility, allowing for more robust and reproducible single molecule measurements under a wider range of experimental conditions. Together, these advancements provide a powerful platform for SMS, bridging the gap between complex experimental data and resulting insights, and significantly contributing to the field of single-molecule biophysics.