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

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 GW͞cm 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.photoswitching ͉ nanoscopy ͉ resolution ͉ saturation ͉ photochromic

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

“…They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .…”

“…They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths.…”

Fluorescence nanoscopy. Methods and applications

“…When this spot of subdiffraction dimensions is scanned across the specimen, a super-resolution image is obtained. Subdiffraction imaging techniques that share this working principle are termed reversible saturable optical transition (RESOLFT) [2]; depending on the particular molecular transition exploited to switch fluorophores to a dark state, the modalities are termed stimulated emission depletion (STED) [3][4][5], ground stated depletion (GSD) [6,7] or, simply, RESOLFT [8].…”

“…Only those fluorophores in this subdiffraction-sized active region can subsequently be excited; hence, fluorescence is assigned unequivocally to that small spot [8,22]. Akin to STED, the size of the subdiffraction spot in RESOLFT imaging is determined by Eq.…”

3‐D stimulated emission depletion microscopy with programmable aberration correction

“…In the past two decades a number of super-resolved microscopy (SRM) techniques have emerged for which the resolution is not limited by diffraction. Following the invention of stimulated emission depletion (STED) microscopy [4,5] and ground state depletion (GSD) microscopy [6] these laser scanning approaches were generalised as "RESOLFT" [7] techniques and were later complemented by stochastically switched single molecule localisation techniques such as PALM [8,9] and STORM [10]. While all these SRM techniques can provide excellent laterally super-resolved images of cells near the microscope coverslip, it is more challenging to realise SRM in the axial direction, particularly inside biological samples that present optical aberrations and scattering.…”