Single-particle tracking fluorescence microscopy (sptFM) is a derivative of single-molecule localization microscopy (SMLM) and offers the possibility to investigate macromolecular motion in life science and food materials. SMLM is a super-resolution optical microscopy methodology characterised by localizing point spread functions (PSFs) originating from single fluorescent molecules, with an accuracy surpassing the diffraction limit of light by roughly one order of magnitude. SptFM tracks single molecules moving through time, describing their position with ~5-40 nm spatial resolution and good temporal resolution. These motions can then be quantitatively characterised and used to reveal macromolecular behaviour.
This thesis aims to advance the field of sptFM by increasing the achievable spatiotemporal resolution, and by increasing the accessibility of the hardware and software in sptFM. Next, sptFM, enriched by these advances, is applied to study dynamic CRISPR-Cas9 behaviour in vivo, and to study the spatiotemporal heterogeneity of κ-carrageenan hydrogels.