(A) Schematic of the light path used for the optical lattice microscope. A collimated circular laser beam is passed through two pairs of cylindrical lenses to illuminate a thin stripe across the width of a ferroelectric spatial light modulator (SLM, Forth Dimension Displays, SXGA-3DM). The remainder of the excitation optical path serves to create a demagnified image of the SLM (81.6 nm pixels) at the focal plane of the excitation objective. First, a lens is used in a 2F configuration to create a diffraction pattern at its front focal plane that is the Fourier transform of the electric field reflected from the SLM. A custom opaque mask with transmissive annuli (Photo-Sciences Inc.) is placed at this plane, and a specific annulus is chosen to remove the unwanted diffraction orders and enforce a limit on the minimum field of view. The electric field transmitted through the mask is then imaged in series onto each of a pair of galvanometers (Cambridge Technology, 6215H) and the rear pupil plane of the excitation objective. The galvanometers serve to translate the light sheet through the specimen in x and z. Finally, the field is reverse transformed by the excitation objective to create the desired lattice light sheet at its front focal plane. The fluorescence generated within the specimen is collected by a detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA, 2 mm WD) whose focal plane is co-incident with the light sheet. Its high NA is essential to maximize the xy resolution and to optimize the light collection for single molecule detection. The excitation objective (Special Optics, 0.65 NA, 3.74 mm WD) was custom designed to fill the remaining available solid angle above the cover slip. A tube lens images the fluorescence from the illuminated slice within the specimen onto a sCMOS camera (Hamamatsu Orca Flash 4.0 v2) capable of frame rates down to 1 ms. A 3D image is produced from a stack of such 2D slices, either by moving the light sheet and detection objective together through the specimen (the former with the z galvo, the latter with a piezoelectric stage [Physik Instrumente, P-621.1CD]) or, far more commonly, by translating the specimen with a second piezo stage through the stationary light sheet along an axis s in the plane of the specimen cover slip. The specimen holder and specimen piezo are mounted on a trio of closed loop micropositioning stages (Physik Instrumente M-663 for horizontal motion in the cover slip plane, M-122.2DD for vertical travel). (B) The XY and XZ point spread function profile of a fluorescent bead (Emission: ∼590 nm; Voxel size, 100 nm in each direction, the radius of the bead is 50 nm). Scale bar, 500 nm. (C) 3D single-molecule localization was performed using 3D Gaussian model (Equation 1) by FISH-QUANT (Mueller et al., 2013). x, y, z localization uncertainty for each 3D localization event was calculated by using the published estimator, Equation 2. The localization histogram was fitted by Extreme Fit by Matlab. The mean and center values were labeled in each plot.