Fig. 1: MINSTED localization principle and spatiotemporal precision.

a, The STED E-PSF (orange) with FWHM d describes the emission probability of a fluorophore (red star) around the center of the donut-shaped STED-beam (red). In MINSTED, the fluorophore experiences the steep edge of the E-PSF (gray sampling region). b, MINSTED localizes by scanning the E-PSF (yellow) in circles around the assumed fluorophore position. Upon detection of each single photon (single photon det.), the circle center is shifted toward the detection position and by that moves (on average) toward the position of the fluorophore (star). c, MINSTED tracks a sudden jump in position (step) of a fluorophore with a few photon detections. Binning the circle centers (rendered for each detection) increases the visibility of the step while compromising temporal information (purple and green lines). Data in a–c are based on simulations using setup parameters. d, Overlaying n = 389 traces (histogram frequency shown as gray scale; STED power, P = 40 mW; E-PSF FWHM, 26 nm; count rate, k = 14 kHz) reveals a nearly exponential response function (blue) with a decay time τ = 1.3 ms and a single-photon spatial precision σ = 3.9 nm. e, Temporal precisions τ (relative standard errors <1% and thus not shown) as a function of the photon count rate \(k\). The arrow indicates the data point representing the data of d. The blue shading indicates the span of minimal (12.9) and maximal (18.8) average number of photons needed for convergence to the new position. The ideal τ (theory) is shown as black dashed line. f, Single-photon precision and step-localization precision (relative standard errors <1‰ and thus not shown) as a function of STED beam power. Excitation powers are given in percentage of approximately 30 µW. The number of steps for each of the 16 conditions is given in Extended Data Table 2.