Decoding the scanning parameters of iterative MINFLUX through Single Particle Tracking

in: European Biophysics Journal with Biophysics Letters (2023)
Vogler, Bela; Reina, Francesco; Zhao, Ziliang; Eggeling, Christian
Detecting the lateral dynamics of molecules on living and model membranes is a challenging field of study, requiring microscopy techniques that can track molecules with single particle level of detail, nanoscopic localization precisions, and with high temporal resolution. MINFLUX microscopy is a recently developed technique that combines concepts of structured illumination and single molecule localization microscopy to achieve unprecedented levels of localization precision. “MINFLUX 3D”, its commercial implementation, relies on several scanning iterations to progressively improve the localization precision of single fluorophores. With this approach, it has been shown how it is possible to obtain single particle trajectories with kHz sampling rates. The iterative scanning procedure, however, raises concerns on whether the technique has any limitation. In particular, we sought to investigate the impact that specific scanning parameters had on the recorded single particle trajectories. We thus investigated the diffusion of single quantum-dot labelled phospholipid analogues on a homogeneous model membrane. The single particle trajectories thus obtained, revealed potential limitations of using iterative MINFLUX scanning for single particle tracking and systematic, unfavorable effects introduced by the scanning parameters. In an effort to theoretically understand possible origins of reported issues, we propose several thought experiments based on fundamental physical and mathematical principles. In conclusion, we highlight possible limiting factors present in the iterative scanning MINFLUX implementation that may advise the growing community in identifying appropriate fields of application.

DOI: Array

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