Volumetric Reconstruction for Improved 3D Single-Molecule Localisation Microscopy

Localisation microscopy provides a dramatic resolution enhancement over conventional microscopy, capable of imaging biological structures at the scale of individual molecules. The image-acquisition speed is however limited by the requirement to time-sequentially image individual fluorescence emissions without significant overlap of the emitter images. For 3D localisation microscopy, each emission is encoded with a spatially extended 3D point-spread function, which further reduces the maximum usable density of emitters in acquired images, and increases the time required to construct a single 3D image. We report a new volumetric localisation microscopy technique that directly deconvolves a single 2D recorded image into a 3D volumetric reconstruction of compact emitter images. Importantly, this enables the domain for detection and fitting of emitter images to be shifted from the acquired, relatively cluttered 2D images to the relatively uncluttered and sparse reconstructed 3D volume.