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.
