Template-free 2D particle fusion in localization microscopy

Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.