![]() First and foremost is the correct identification of single molecules which is often confounded by repeated cycling between on-off states, resulting in multiple detections from a single emitter 11, 12, 13. SMLM techniques are potentially potent quantitative tools, however single molecule imaging and quantification have significant caveats which are critical for accurate image acquisition, reconstruction, and quantification. Since the early inception of these techniques, a number of variations have been developed which aim to address the shortcomings of previous iterations and improve on both spatial resolution and the quantitative capability of SMLM (including iPALM, PAINT, and MINFLUX) 8, 9, 10. While in STORM paired dyes or organic fluorophores capable of switching within an appropriate redox buffer (dSTORM) are used, PALM relies on photoswitching fluorescent proteins (e.g. The key difference between these two approaches lies in the nature of the reporters used to generate ‘blinking’ events which result from the stochastic activation of fluorophores. PALM (Photo Activated Localisation Microscopy) and STORM (Stochastic Optical Reconstruction Microscopy) are archetypal SMLM methods which are based on the stochastic activation of subsets of molecules within a sample to allow for precise localisation and rendering post-acquisition 2, 3. ![]() Of the many super-resolution methods currently available, single molecule localisation microscopy (SMLM) is unique in that it not only offers spatial resolution in the tens of nanometers 1, 2, 3, 4, 5, 6, but a means of quantifying single emitters for insights into protein stoichiometry and organisation 2, 3, 4, 7. Super-resolution imaging techniques offer unique insights into cellular structures and organisations on a nanoscale inaccessible through conventional light microscopy. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy.
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