Single molecule localisation microscopy image using Nse4-HaloTag budding yeast and JFX650 fluorophore

Single molecule localisation microscopy (SMLM) image dataset using Nse4-HaloTag budding yeast and JFX650 fluorophore. <br> Live <em>S. cerevisiae</em> cells expressing Nse4 fused to the self-labelling HaloTag (previously described in Hallett <em>et al</em> (2022), DOI: 10.1093/nar/gkac692) were grown in synthetic complete media until they reached mid-log phase. JFX650 HaloTag ligand (a gift from Luke Lavis, Janelia Research Campus, USA) was added to the culture to a final concentration of 50nM. Cells were incubated in the presence of the dye for 20 min before being harvested, washed in fresh media and deposited on an agarose pad (1% w/v agarose, 50% v/v OptiPrep, 50% v/v 2× SC-media). Samples were then mounted on a circular coverslip and imaged on a custom-built microscope (previously described in Etheridge <em>et al</em> (2021), DOI 10.7554/eLife.68579). Movies were recorded with an exposure time of 10 ms per frame and continuous illumination with a 642nm laser. JFX650 molecules were photo-converted back from a long-lived dark-state by illumination with a pulse of a 488 nm laser. The camera was a Photometrics Evolve Delta using: 45 counts/photon total gain; 0.3017 counts read noise; 400 counts offset. Optical magnification creates 101nm pixels. <br> Nse4 is a sub-unit of a DNA binding protein complex SMC5/6 known for its roles in DNA repair. The individual molecules observed in the image may be diffusing, or immobile when bound to the DNA. <br> The dataset includes: <br> BuddingYeast_Nse4-Halo_10ms.tif: SMLM image stack (10ms per frame) BuddingYeast_Nse4-Halo_Brightfield.tif: A brightfield image of the yeast cells BuddingYeast_Nse4-Halo_10ms_Fit_Template.txt: An example template for fitting the localisations using the Genome Damage and Stability Centre (GDSC) SMLM ImageJ plugin Peak Fit BuddingYeast_Nse4-Halo_10ms.tif.results.xls: An example results file containing the fitted localisations BuddingYeast_Nse4-Halo_10ms.tif.exported.csv: An example of the results exported in a custom comma-separated values (CSV) format (intensity in photons; distances in nm) <br> <br>