Parameter optimization for MINFLUX microscopy enabled single particle tracking

MINFLUX fluorescence microscopy is a recently introduced super-resolution approach for studying cellular structures and their dynamics with highest detail. MINFLUX (MFX) performs Single Particle Tracking (SPT) at runtime. The ad hoc signal interpretation necessary to sustain the method relies on several parameters, which need to be optimized in relation to the sample under study, such as fluorescent lipid analogs in membranes, to ensure the fidelity of the measurement. We propose a parameter optimization strategy, give an overview of the most important parameters, present a theoretical upper limit for trackable diffusion rates, and demonstrate MFX-enabled SPT of fast $\left(\left(D_{\mathrm{MSD}}\right)=2.5\mu m^2/s\right)$ lateral Brownian motion of lipids in membranes.