Fig. 1: Optical setup and tracking concept.

a Illustration of the optical setup, laser excitation path with amplitude modulation, spatial light modulator (SLM) for PSF engineering, and electro-optic deflectors (EOD). Following the excitation light path after the dichroic mirror, a piezo-driven tip/tilt mirror scanner is used for long-range scanning, the light path ends with an objective and sample. Collected fluorescence is de-scanned by the tip/tilt mirror and passed through the dichroic mirror, fluorescent filters, and ends with a confocal detection based on single photon counting avalanche photodiodes. b Tracking pattern created by the EODʼs . On the left is a view of the pattern from the top, and on the right viewed from the side. Stars indicate the placement of the Gaussian focal points. c Graphical representation of the real-time and data post-processing. Real-time; The PSF is shaped at the back focal plane (BFP) so that the desired PSF appears at the focal plane (FP). The center part of the PSF is selected by a 2 μm circular aperture, the tracking pattern is created by moving, using the EODʼs, desired part of the PSF into the center area where the reporter is exposed, simultaneously the aperture blocks the remaining part of the PSF. Emitted photons are detected and processed by the field-programmable gate array (FPGA) to obtain a new piezo position. Post-processing (left to right); the PSF is sampled as a z-stack which is interpolated, and processed with the EOD pattern and piezo center. Along with the photon counts, this fully reconstructs the illumination pattern and gives the parts necessary for constructing the likelihood.