Fig. 1: Encoding the 3D position of single-molecule fluorescence into a 2D image.

ai The standard (2D) point spread function (PSF) can be modified to encode 3D position by phase modulation in the back focal plane (BFP, indicated by gray level) of a widefield microscope. aii Key 3D SMLM techniques include astigmatism, the double helix PSF, and the tetrapod PSF, shown here in an 8 × 8 μm² field-of-view (scale bars are 1 μm). aiii The associated PSF footprints integrated over their entire axial range (color-coded by depth). The loss in lateral resolution at the expense of axial range leads to a lower signal-to-noise ratio. b Schematic of the microlens array used in this SMLFM platform, the PSF in the central perspective view, and the PSF footprint integrated over the entire 8 μm axial range (color-coded by depth). A simplified optical diagram of SMLFM is also shown on the right, where OBJ = objective, TL = tube lens, FL = Fourier lens, and MLA = microlens array. Optical diagrams for all the 3D techniques discussed herein can be found in Supplementary Fig. 2. Pixel size is 110 nm for standard, astigmatism and the tetrapod PSF, and 266 nm for the DHPSF and SMLFM to reflect experimental parameters.