Abstract
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength that also excites fluorescence. Positive switching RSFPs have a reversed light response, where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. On the basis of the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-states of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential illumination steps.
Highlights
Nanoscopy, or diffraction-unlimited super-resolution fluorescence microscopy, enables the visualization of cellular structures at the nanoscale
Most implementations of RESOLFT nanoscopy rely on reversibly switchable fluorescent proteins (RSFPs), which belong to the group of GFP-like fluorescent proteins.[8,9]
Kohinoor has been used for a demonstration of point-scanning RESOLFT nanoscopy, but we found that Kohinoor is still prone to switching fatigue
Summary
Diffraction-unlimited super-resolution fluorescence microscopy, enables the visualization of cellular structures at the nanoscale. RESOLFT (reversible saturable optical linear (fluorescence) transitions) nanoscopy is a coordinate-targeted approach that relies on reversibly switchable fluorophores.[4−7] In pointscanning RESOLFT nanoscopy, a single laser beam creating a doughnut-shaped intensity distribution with a zero at its center is used to transfer molecules into a nonfluorescent offstate. Most implementations of RESOLFT nanoscopy rely on reversibly switchable fluorescent proteins (RSFPs), which belong to the group of GFP-like fluorescent proteins.[8,9] These proteins feature a β-barrel structure with a central α-helix containing the autocatalytically formed chromophore.[10] RSFPs can be reversibly toggled between a fluorescent on- and a nonfluorescent off-state by illumination with two different
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