Parameter Optimization for Iterative 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. Iterative MINFLUX (iMFX) 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 analogues in membranes, to ensure the fidelity of the measurement. We propose a parameter optimization strategy, an overview of the most important parameters, present a theoretical upper limit for trackable diffusion rates, and demonstrate iMFX-enabled SPT of fast (⟨DMSD⟩ = 2.5μm2/s) lateral Brownian motion of lipids in membranes.