Improving localization precision via restricting biomolecule confined stochastic motion in SMLM

Abstract Single-molecule localization microscopy (SMLM) boosts its applications when combined with the studies of cells, in which nanometer-sized biomolecules are irresolvable due to diffraction limit unless being subjected to SMLM. Although being well immobilized, given the nanometer sizes of biological molecules, they are still capable of movement stochastically around their immobilized sites. The influence of such motion on image quality and possible improvements have not yet been systematically investigated. Here, we accessed the biomolecule stochastic motion in SMLM by calculating the displacements between different localizations from the same molecule in single-molecule samples of Alexa Fluor-647-conjugated oligonucleotides. We found that, for most molecules, localization displacements at random frame intervals are remarkably larger than those between temporally neighbouring frames despite of drift correction, showing that biomolecule stochastic motion is involved in SMLM. Furthermore, the localization displacements were observed to increase with frame intervals and then saturate, suggesting biomolecule stochastic motion is confined within a finite area. Moreover, we showed that the localization precision is deteriorated by enlarging molecule sizes and improved by sample post-fixation. This study reveals confined stochastic motion of biomolecules increase localization uncertainty in SMLM, and improved localization precision can be achieved via restricting biomolecule stochastic motion.