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.

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

The exquisite selectivity, sensitivity, and spatial resolution obtained with fluorescence spectroscopy and imaging have led to an ever-increasing number of applications. With the development of detectors approaching 100 % quantum efficiencies and sophisticated collection optics, the bottleneck of current fluorescence microscopy is the fluorophores used, which pose severe limitations owing to photobleaching and blinking. Most of the basic dye structures that are currently used in fluorescence microscopy have been known since their use in the development of dye lasers.[1] Increasing demands posed by fluorescence microscopy and single-molecule and high-resolution applications [2,3] have spurred the development of new kinds of emitters such as semiconductor nanocrystals, silver nanoclusters, and new derivatives of fluorescent proteins.[4] In comparison, the advancement of classical organic dyes such as rhodamine or cyanine derivatives has been incremental despite some progress with regard to labeling chemistry, solubility in water, and the availability of bright and photostable near-IR dyes. Approaches for their improvement comprise increasing brightness by multichromophore systems, intramolecular triplet quenching, and decreasing the sensibility for reactions with singlet oxygen. [5] For different reasons, none of these approaches has been implemented with great success in fluorescence microscopy.Here we present a new approach to minimize photobleaching and blinking by recovering reactive intermediates. The method is based on the removal of oxygen and quenching of triplet as well as charge-separated states by electrontransfer reactions. For this reason, a structure that contains reducing as well as oxidizing agents, that is, a reducing and oxidizing system (ROXS) is used. The success of the approach is demonstrated by single-molecule fluorescence spectroscopy of oligonucleotides labeled with different fluorophores, that is, cyanines, (carbo-)rhodamines, and oxazines, in aqueous solvents; individual fluorophores can be observed for minutes under moderate excitation with increased fluorescence brightness. Thermodynamic considerations of the underlying redox reactions support the model, yielding a comprehensive picture of blinking and photobleaching of organic fluorophores.Typically, the photophysics of fluorophores is described by a three-state model including the ground and first excited singlet states, S 0 and S 1 , respectively, and the lowest triplet state T 1 . Owing to its longer lifetime, T 1 is considered to be the photochemically most active state. Quenching of T 1 by molecular oxygen, for example, can generate reactive singlet oxygen, and therefore oxygen is removed in demanding applications, for example, with the aid of an enzymatic oxygen-scavenging system.[6] The disadvantage of oxygen removal, however, is the increase of the triplet state lifetime with negative effects for the brightness of the fluorophore and increased probability for other follow-up reactions from the triplet state. Alternatively, redu...

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher beta-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of >90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.