'n Ondersoek na enkeldeeltjie-nasporingsmetodes vir fotosintetiese komplekse

Abstract

Photosynthesis is the direct source of energy for life on Earth. This process begins with the absorption of photons by light harvesting complexes, such as light har- vesting complex II (LHCII) in plants and phycobilisome in cyanobacteria. These systems’ combination of complex, individual behaviour and high photoactivity make it a good target for single-molecule spectroscopy (SMS). However, standard SMS approaches utilise an environment that badly resembles the natural one. In particular, a particle is typically isolated and attached to a surface. A technique that is capable of keeping a particle in the detection volume without disturbing the surrounding environment is real-time single particle tracking (SPT). In this disser- tation, the underlying theory around SPT is investigated and applied. Preliminary experimental development and testing are also described. Statistical modeling and dynamic simulations were used to compare the theoretical performance of different methods. The orbital, knight’s tour and MINFLUX methods were compared, and both fluorescence-based and iSCAT approaches were investigated. It was found that the knight’s tour method can track the fastest diffusion, while MINFLUX has the best precision, albeit only for slow particles. To compare iSCAT and fluorescence, various biological examples were considered, including intrinsically fluorescing particles and particles with a fluorescent label. The relative success of iSCAT compared to fluorescence is dependent on the particle size, photophysical properties of the fluorophore, and the fluorophore density. The extension of an SMS setup for single-particle tracking is also described. This includes hardware and software development, as well as preliminary testing and measurements. Future prospects are also discussed.