Imaging force-dependent cytoskeletal dynamics in tumour cells using single-molecule localisation microscopy

Single-molecule localisation microscopy (SMLM) separates fluorophore emissions in time to create a tenfold increase in spatial resolution along all three axes. However, long acquisition times make live-cell imaging difficult. These can only be shortened by acquiring 'high-density' data, which increases the risk of generating artificial structures in the final image - a risk that is further increased when imaging in three dimensions. The Haar Wavelet Kernel software (HAWK) is designed to remove artefacts during high-density imaging, and we are using the technology to develop a HAWK Method of Assessment (HAWKMAN) for SMLM images. Using a variety of point-spread function asymmetries, we have also been running evaluations of HAWK's propensity for handling different types of 3D data. Future work will see these technologies applied to live-cell, 3D SMLM imaging using selective plane illumination microscopy to study cancer cell motility mechanics in 3D environments over short timescales.