Fig. 1: OLS provides near full-field homogeneous illumination enabling SMT.

a, OLS is a single-objective, light-sheet imaging modality that provides illumination homogeneity, a large achievable FOV and superior background fluorescence rejection. OLS is suitable for SMLM and SMT measurements. Here the SMT setup relies on the Halo-tagging of protein targets of interest. Note that molecules emitting isotropically, located within the emission cone and constrained by the rolling shutter width, contribute to the photons on the camera. b, JF₅₄₉ or JF₆₄₆ organic fluorophores are used to provide individual PSFs with appropriate signal for detection to conduct frame-to-frame linking and trajectory generation. From these coordinates and trajectories, a variety of metrics including protein diffusion and spatial localization can be extracted. Middle: regions of interest displaying the detection and tracking of two PSFs of interest. Blue circles denote PSFs. Right: trajectories rendered for all four frames. Dotted rectangle box denotes the smaller region of interest c, Representative sampling areas imaged with OLS illumination from Halo-KEAP1-expressing U2OS cells. Trajectories are plotted across a 1.5-s acquisition and color coded based on the measured diffusion coefficient with nuclear mask outlines overlaid with a black dotted line and cell boundaries with a gray solid line. Left: full 250 × 190 μm² OLS FOV. Right: zoomed-in region centered around one cell. Note that a typical FOV consisting of U2OS cells seeded at 6,000 cells per well contains 40–50 cells. d, Representative average spatial SNR maps per pixel calculated across 1,232 FOVs for a 384-well plate imaged for each of HILO and OLS. Inset: HILO-sized image. e, OLS-generated dose–response curves of the KEAP1 inhibitor KI-696 on U2OS cells expressing halo-tagged KEAP1. Data were acquired across four microscopes and imaged on our high-throughput SMT platform. 72 FOVs from 12 wells were captured for each KI-696 concentration and DMSO negative control, on 6–7 spatially randomized 384-well plates. Error bars denote the s.d. f, Representative images of FOV size for each of five frame rates in the range of 100–1,250 Hz. Trajectories are overlaid onto a mean projection for the Hoechst channel (blue) and colored by their maximum likelihood diffusion coefficients. The number of FOV replicates per frame rate were: n = 88 (100 Hz), n = 88 (200 Hz), n = 132 (400 Hz), n = 198 (800 Hz) and n = 264 (1,250 Hz). g, Mean trajectory length plotted as a function of frame rate. Track length is defined as the number of spots linked per trajectory. h, Fraction of trajectories with a diffusion coefficient > 10 μm² s⁻¹ as a function of frame rate for DMSO and KI-696-treated cells, computed from the state array posterior mean occupations. n = 88 (100 Hz), n = 88 (200 Hz), n = 132 (400 Hz), n = 198 (800 Hz) and n = 264 (1,250 Hz) with each FOV containing approximately 40–50 cells at 100 Hz. i, Accuracy of state profile recovery from optical–dynamical simulations of SMT across several frame rates for three distinct state mixtures. Error bars denote the s.d.