Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein Dreiklang.

Diffraction-unlimited far-field super-resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending on whether this is induced in a spatially controlled manner using a pattern of light (coordinate-targeted) or stochastically on a single-molecule basis, specific requirements on the fluorophores are imposed. Therefore, the fluorophores are usually utilized just for one class of methods only. In this study we demonstrate that the reversibly switchable fluorescent protein Dreiklang enables live-cell recordings in both spatially controlled and stochastic modes. We show that the Dreiklang chromophore entails three different light-induced switching mechanisms, namely a reversible photochemical one, off-switching by stimulated emission, and a reversible transfer to a long-lived dark state from the S1 state, all of which can be utilized to overcome the diffraction barrier. We also find that for the single-molecule-based stochastic GSDIM approach (ground-state depletion followed by individual molecule return), Dreiklang provides a larger number of on-off localization events as compared to its progenitor Citrine. Altogether, Dreiklang is a versatile probe for essentially all popular forms of live-cell fluorescence nanoscopy.

rsEGFP2 enables fast RESOLFT nanoscopy of living cells.

The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25-250 times faster recordings.DOI:http://dx.doi.org/10.7554/eLife.00248.001.

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

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

Dual-label STED nanoscopy of living cells using photochromism.

We demonstrate live-cell STED microscopy of two protein species using photochromic green fluorescent proteins as markers. The reversible photoswitching of two markers is implemented so that they can be discerned with a single excitation and STED wavelength and a single detection channel. Dual-label STED microscopy is shown in living mammalian cells.

Molecular basis of the light-driven switching of the photochromic fluorescent protein Padron.

Reversibly switchable fluorescent proteins can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with the light of two different wavelengths. The molecular basis of the switching process remains a controversial topic. Padron0.9 is a reversibly switchable fluorescent protein with "positive" switching characteristics, exhibiting excellent spectroscopic properties. Its chromophore is formed by the amino acids Cys-Tyr-Gly. We obtained high resolution x-ray structures of Padron0.9 in both the fluorescent and the nonfluorescent states and used the structural information for molecular dynamics simulations. We found that in Padron0.9 the chromophore undergoes a cis-trans isomerization upon photoswitching. The molecular dynamics simulations clarified the protonation states of the amino acid residues within the chromophore pocket that influence the protonation state of the chromophore. We conclude that a light driven cis-trans isomerization of the chromophore appears to be the fundamental switching mechanism in all photochromic fluorescent proteins known to date. Distinct absorption cross-sections for the switching wavelengths in the fluorescent and the nonfluorescent state are not essential for efficient photochromism in fluorescent proteins, although they may facilitate the switching process.

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica.

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.