Optimized MINFLUX microscopy for reliable high-speed 2D/3D single-particle tracking

MINFLUX microscopy enables nanometer-precision localization and real-time single-particle tracking (SPT) using iterative structured excitation. While this permits two- (2D) and threedimensional (3D) tracking at kilohertz rates, reliable performance critically depends on sequence parameters and hardware latency. Here, we present a control-theoretic description of MINFLUX-enabled SPT and derive analytical expressions for the time-to-localization in 2D and 3D. From this framework, we establish an upper theoretical limit for trackable diffusion coefficients and identify the effective localization range as a fundamental constraint. Our analysis demonstrates that tracking reliability is governed primarily by acquisition speed rather than photon-limited precision, motivating a speed-first optimization strategy. These guidelines expand the applicability of MINFLUX to high-speed, high-fidelity tracking in biological systems.