Accelerated MINFLUX Nanoscopy, through Spontaneously Fast‐Blinking Fluorophores. Issue 12 (15th January 2023) - Remmel, Michael
Abstract: The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized for a high photon yield and rather long on‐times (tens of milliseconds). Here, accelerated MINFLUX nanoscopy with up to a 30‐fold gain in localization speed is presented. The improvement is attained by designing spontaneously blinking fluorescent markers with remarkably fast on‐times, down to 1–3 ms, matching the iterative localization process used in a MINFLUX microscope. This design utilizes a silicon rhodamine amide core, shifting the spirocyclization equilibrium toward an uncharged closed form at physiological conditions and imparting intact live cell permeability, modified with a fused (benzo)thiophene spirolactam fragment. The best candidate for MINFLUX microscopy (also suitable for STORM imaging) is selected through detailed characterization of the blinking behavior of single fluorophores, bound to different protein tags. Finally, optimization of the localization routines, customized to the fast blinking times, renders a significant speed improvement on a commercial MINFLUX microscope. Abstract : MINFLUX nanoscopy with a 30‐fold localization speed increase. Rather than adapting the single‐molecule localization process to commonly used fluorescent markers with long on‐times (tens of milliseconds), we designed spontaneously blinking fluorophores with on‐times in the millisecond time‐range. The achieved synergy between the localization and blinking processes results in up to 30× localization speed gain, while retaining biomolecular‐sized spatial resolution.