Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

TLDR

Fluorescence microscopy is essential but diffraction limits its resolution; previous super‑resolution methods use stimulated emission depletion with high‑intensity picosecond lasers. The study demonstrates that the diffraction barrier can be surpassed using illumination eight orders of magnitude lower than conventional methods. Resolution is achieved by reversible photoswitching of the protein asFP595 between fluorescent and dark states driven by a spatial intensity pattern with a zero, enabling RESOLFT to resolve 50–100 nm. The achieved 50–100 nm resolution is limited only by the protein’s photokinetics and the quality of the intensity zero, indicating that further optimization could reach true molecular resolution.

Abstract

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm 2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50–100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.