Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.

Adlerauge: dSTORM nutzt das reversible Schalten von konventionellen Fluoreszenzfarbstoffen bei der Bestrahlung mit Licht zweier Wellenlängen, um hochaufgelöste Bilder zu erhalten. Die elegante und einfache Methode kann zelluläre Strukturen mit einer Aufösung von ca. 20 nm ohne Aktivatorfarbstoff deutlich unterhalb der Auflösungsgrenze sichtbar machen.

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.

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.

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