Analysing errors in single-molecule localisation microscopy

In single molecule localisation microscopy (SMLM) a super-resolution image of the distribution of fluorophores in the sample is built up from the localised positions of many individual molecules. It has become widely used due to its experimental simplicity and the high resolution that can be achieved. However, the factors which limit resolution in a reconstructed image, and the artefacts which can be present, are completely different to those present in standard fluorescent microscopy techniques. Artefacts may be difficult for users to identify, particularly as they can cause images to appear falsely sharp, an effect called artificial sharpening. Here we discuss the different sources of error and bias in SMLM, and the methods available for avoiding or detecting them.