Super-resolution microscopy is a relatively new and rapidly growing field. Development has been largely technology-driven, with high power lasers, higher resolution CCD cameras, and increasing computing power all enabling new biological questions to be explored. Single-molecule imaging is the tool of choice for studying systems where heterogeneity is present; ensemble methods can average away the interesting behaviour and lead to false conclusions. This thesis develops and optimises bespoke fluorescence microscopy for application to three biological questions, each pushing a limit of super-resolution imaging.
Super-resolution imaging of lambda DNA labelled with the intercalating dye YOYO-1 and the minor groove binder SYTO-13 at localisation precisions of 40nm and 62nm respectively has been achieved in preparation for combined fluorescence imaging and magneto-optical tweezers experiments. The combination of these two methods is challenging as both operate with low tolerances.\
Single-molecule tracking was used to measure the diffusion coefficients of the chemokines CXCL13 and CCL19 at extremely high temporal resolution. Single-molecule imaging was found to have advantages over the ensemble techniques of FRAP and FCS for measuring the diffusion coefficient of the test molecule; Alexa Fluor 647 labelled bovine serum albumin. The diffusion coefficients of the two chemokines, CXCL13 and CCL19 were found by single particle tracking at sub-millisecond timescales in a collagen matrix to be 6.2±0.3µm2s-1 and 8.4±0.2µm2s-1. Further, CXCL13 was tracked in B cell follicle regions of ex vivo lymph node tissue sections at ~2 millisecond timescales, giving a diffusion coefficient of 6.6±0.4µm2s-1.\
Fluorescence microscopy was used to elucidate the stoichiometry of YOYO-1 on DNA origami tiles after treatment with low temperature plasma. Undamaged tiles were found to have a mean stoichiometry of 67.4±25.2 YOYO-1 molecules and a model of LTP damage to DNA origami tiles was proposed.
