Fig. 1.

MINFLUX probes the position of an emitter with light distributions featuring intensity zeros that are targeted to defined coordinates in sample space. (A) Schematic of the MINFLUX setup used here. A laser beam is structured by a phase mask to obtain a doughnut-shaped excitation profile $I\left(\overline{r}\right)$ at the focal plane of the objective. The emitter fluorescence is collected in backscattering geometry and separated from the excitation beam by a dichroic mirror (DM). After passing a bandpass filter (BPF), the fluorescence photons are focused onto a confocal pinhole (PH) and counted by a detector (Det.). A field-programmable gate array (FPGA) board controls the deflection system, modulates the beam intensity, and processes the detected photons. (B) (Upper) The intensity zero of the doughnut is targeted in quick succession to coordinates ${\overline{r}}_{b0},\dots ,{\overline{r}}_{b3}$ lying within a circle of diameter $L$, defining the set of targeted coordinates. (Lower) Example of a collected photon trace ($n_0,\dots ,n_3$) for a molecule transiting the rapidly retargeted doughnut beams. (C) Visualization of the position-dependent localization uncertainty. The blue ellipses represent the $e^{-1/2}$ contour level of the covariance matrix ${\mathrm{\Sigma }}_{\mathrm{CRB}}\left(\overline{r}\right)$ as a quadratic form, for a total of N = 1,000 photons and $L=50$ nm. The red encircled area defines a region of interest (ROI). (D) Optimal $L$ value $L_{\mathrm{opt}}$ (black) for two SBR values ($\mathrm{SBR}=\infty ,\hspace{0.17em}\mathrm{SB}{\mathrm{R}}_{L=50\mathrm{nm}}^{\overline{r}=\overline{0}}=12$) and corresponding MINFLUX CRB ${\sigma }_{\mathrm{CRB}}$ (blue bands, $L=L_{\mathrm{opt}}$, N = 100 photons) as a function of the diameter of the ROI. A considerable improvement over ideal camera performance is achievable especially for small ROIs. In the presence of background $L_{\mathrm{opt}}$ saturates for small ROI values. (E) Localization precision as a function of time resolution. The MINFLUX CRB ${\sigma }_{\mathrm{CRB}}$ in a 30-nm ROI (blue band, $L=50$ nm, $\gamma =350$ kHz, $\mathrm{SB}{\mathrm{R}}_{L=50\mathrm{nm}}^{\overline{r}=\overline{0}}=12$) is compared with ideal camera performance without background (black) and with realistic background contributions (dashed black, ${\text{SBR}}_{\text{c}}=18$).