Abstract
Super-resolution fluorescence imaging using single molecule localization microscopy (SMLM) can provide spatial resolution higher than the one imposed by diffraction limit. This virtue has made SMLM a powerful imaging tool for biomedical research. But this feature comes at the cost of having to acquire several thousands of images of the same cell, over measurements times ranging from tens of minutes to hours. Over such long data acquisition times, sample drift becomes a critical problem reducing resolution of the reconstructed image and cannot be neglected. We present here a simple method to prevent long-term drift using periodic registration to bright-field reference image of the cell at the desired focal plane. We combined this with a post-collection correction based on nearest-neighbor distributions that removes residual drift that happens while collecting data between registrations and corrects small remaining registration errors beyond the limit of the bright-field registration. This hardware/software combination does not require any specialized hardware or fiducials. We demonstrate robustness of this approach in 2D and 3D using DNA-PAINT imaging of several cellular structures in fixed cells.
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