Motion screening of fiducial marker for improved localization precision and resolution in SMLM.

Single-molecule localization microscopy (SMLM) provides unmatched high resolution but relies on accurate drift correction due to the long acquisition time for each field of view. A popular drift correction is implemented via referencing to fiducial markers that are assumed to be firmly immobilized and remain stationary relative to the imaged sample. However, there is so far lack of efficient approaches for evaluating other motions except sample drifting of immobilized markers and for addressing their potential impacts on images. Here, we developed a new approach for quantitatively assessing the motions of fiducial markers relative to the sample via mean squared displacement (MSD) analysis. Our findings revealed that over 90% of immobilized fluorescent beads in the SMLM imaging buffer exhibited higher MSDs compared to stationary beads in dry samples and displayed varying degrees of wobbling relative to the imaged field. By excluding extremely high-MSD beads in each field from drift correction, we optimized drift correction and experimentally measured localization precision. In SMLM experiments of cellular microtubules, we also found that including only relatively low-MSD beads for drift correction significantly improved the image resolution and quality. Our study presents a simple and effective approach to assess the potential relative motions of fiducial markers and emphasizes the importance of pre-screening fiducial markers for improved image quality and resolution in SMLM imaging.