Abstract
I will show how an in-depth description of the basic principles of diffraction-unlimited fluorescence microscopy has spawned MINFLUX [1-4], a recent superresolution method that has reached the resolution of the size of a fluorophore molecule. Providing 1–3 nanometer resolution in fixed and living cells, as well as localization precisions in the Angström range, MINFLUX and the related MINSTED concept [5,6] are being established for routine applications in the biomedical sciences [4]. Relying on fewer fluorescence photons than other methods, these techniques are also poised to characterize dynamic processes at the single protein level, as already demonstrated by tracking sub(nanometer) details of the unhindered stepping of the motor protein kinesin-1 on microtubules at up to physiological ATP concentrations [7].
[1] Balzarotti, F., Eilers, Y., Gwosch, K. C., Gynnå, A. H., Westphal, V., Stefani, F. D., Elf, J., Hell, S.W. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606-612 (2017).
[2] Eilers, Y., Ta, H., Gwosch, K. C., Balzarotti, F., Hell, S. W. MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution. PNAS 115, 6117-6122 (2018).
[3] Gwosch, K. C., Pape, J. K., Balzarotti, F., Hoess, P., Ellenberg, J., Ries, J., Hell, S. W. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).
[4] Schmidt, R., Weihs, T., Wurm, C. A., Jansen, I., Rehman, J., Sahl, S. J., Hell, S. W. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat. Commun. 12:1478.
[5] Weber, M., Leutenegger, M., Stoldt, S., Jakobs, S., Mihaila, T. S., Butkevich, A. N., Hell, S. W. MINSTED fluorescence localization and nanoscopy. Nat. Photon. 15, 361-366 (2021).
[6] Weber, M., von der Emde, H., Leutenegger, M., Gunkel, P., Sambandan, S., Khan, T. A., Keller-Findeisen, J., Cordes, V. C., Hell, S.W. MINSTED nanoscopy enters the Ångström localization range. Nat. Biotechnol., 41, 569-576 (2023).
[7] Wolff, J.O., Scheiderer, L., Engehard, T., Engelhardt, J., Matthias, J., Hell, S.W. MINFLUX dissects the unimpeded walking of kinesin-1, Science, 379, 1004-1010 (2023).