MINFLUX tracking for real-time observation of biological processes at molecular resolution

Session

Poster Session

Authors

Evelyn Garlick (1), Clara-Marie Gürth (1), Ulf Matti (1), Tobias Engelhard (1), Isabelle Jansen (1), Roman Schmidt (1), Tobias Weihs (1), Joachim Fischer (1), Christian Wurm (1)

Affiliations

1. Abberior Instruments

Keywords

MINFLUX, single particle tracking, single molecule localization, microscopy, 2D, 3D, super resolution, nanoscopy

Abstract text

Intracellular processes occur on the molecular level and at various speeds. For a long time, microscopy methods for visualizing dynamic processes were limited to either a high spatial resolution or high acquisition speed, while often exposing the sample to high light intensities.

MINFLUX nanoscopy has paved the way towards investigating the dynamics of (macro)molecular complexes and machines on a nanoscopic level. In MINFLUX, the localization of single fluorophores is accomplished with a minimal number of photons. By tailoring the probing scheme to the dimensions and time scales of the individual experiment, it is possible to collect thousands of data points from a single fluorophore, with a temporal resolution of up to 100 µs and track lengths of several seconds. Thus, MINFLUX allows to track single fluorophores at so far unmatched spatiotemporal resolution. 

Figure 1. Tracking of a single abberior STAR RED fluorophore in the cell membrane of a living HeLa cell with 3691 datapoints over the time span of 0.570 seconds (blue to red).

While alternative approaches often lack molecular specificity or live-cell compatibility, MINFLUX combines single-digit nanometer localization precision with a standard light microscopy setup, allowing to easily implement this technique into common biological workflows in a wide range of applications. We demonstrate MINFLUX as a versatile tool to track single biological molecules in 2D or 3D, in vitro and in living cells at molecular resolution. In addition, we envision and test the capability and potential of parallel two-color tracking with MINFLUX.

References

[1] Balzarotti, et al. (2017): Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science, 355, 606-612

[2] Schmidt, et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat Commun. 2021 Mar 5;12(1):1478

[3] Deguchi, et al. (2022) Direct observation of motor protein stepping in living cells using MINFLUX. Science, 2023 Mar 10;379(6636):1010-1015