Localization of fluorophores is key for optical measurements at the nanoscale and beyond ensemble averaging. Prominent examples are single-molecule tracking and super-resolution imaging by single-molecule localization microscopy (SMLM). Among single-molecule localization methods, MINFLUX outstands for achieving a ~10-fold improvement in localization precision over the most popular wide-field camera-based approaches, achieving 1 nm resolution with common fluorophores under ambient conditions. However, its widespread application has been hindered due to its high technical complexity, elevated cost, and, we believe, because it was originally presented in a conceptual framework that may be difficult to grasp at first sight. Here, we will present a new conceptual framework to explain MINFLUX and other related methods such as orbital tracking, and a new method, RASTMIN, that delivers localization precision equivalent to MINFLUX and can be implemented on any scanning (confocal) fluorescence microscope.

References:

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynma, V. Westphal, F. D. Stefani, J. Elf, S. W. Hell
“Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes”
Science 355 (2017) 606-612

L. A. Masullo, F. Steiner, J. Zähringer, L. F. Lopez, J. Bohlen, L. Richter, F. Cole, P. Tinnefeld, F. D. Stefani
“Pulsed Interleaved MINFLUX”
Nano Letters 21 (2021) 840-846

L. A. Masullo, L. F. Lopez, F. D. Stefani
“A common framework for single-molecule localization using sequential structured illumination”
Biophysical Reports 2 (2022) 100036

L. A. Masullo, F. D. Stefani
“Multiphoton single-molecule localization by sequential excitation with light minima”
Light: Science & Applications 11 (2022) 70

L. A. Masullo, A. M. Szalai, L. F. Lopez, M. Pilo-Pais, G. P. Acuna, F. D. Stefani
“An alternative to MINFLUX that enables nanometre resolution in a confocal microscope”
Light: Science & Applications (2022) – just accepted