Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes.

We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated π-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.

In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET.

Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology.

Spiropyrans as molecular optical switches.

Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.

Fluorescent proteins for single-molecule fluorescence applications.

We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications.