Pinpointing macromolecular motion: Single-particle tracking fluorescence microscopy advances and applications

Macromolecular motion is dictated by diffusion on >100 nm spatial scales, while specific interactions occur on the nanoscale based on Van der Waals, electrostatic, and hydrophobic/hydrophilic forces. Characterising macromolecular motion reveals fundamental insights when relating diffusional behaviour to the function of a macromolecule of interest. To adequately investigate diffusion, a technique is required that ideally 1) minimises sample invasion and destruction; 2) minimises spatiotemporal averaging; 3) reaches molecular specificity; 4) is accessible by non-experts of the technique; and 5) has a spatiotemporal resolution of at least ∼5-100 nm and ∼10 ms, and preferably even better.

Single-particle tracking fluorescence microscopy (sptFM) is a derivative of single-molecule localization microscopy (SMLM) and offers the possibility to investigate macromolecular motion while upholding these criteria. 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. While sptFM is a promising technique to observe macromolecular motion, it can be further improved. SptFM can then be applied to study macromolecular diffusion in life science and soft matter.

This thesis aims to advance the field of sptFM by increasing the achievable spatiotemporal resolution, and by increasing the accessibility of the hardware (miCube microscopy platform) and software (phasor-based single molecule localization microscopy or pSMLM) 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.