3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

•Routine testing and calibration are essential for high quality super-resolution imaging.•We present a number of time and cost effective DNA-PAINT based test samples that have good stability.•We demonstrate procedures for validating SMLM 3D system performance.•We describe a procedure to establish and test quantitative analysis of DNA-PAINT data. Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.