High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm −2 ) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm −2 ) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm −2 ) in dense cytosolic tubulin datasets.