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.

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.

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.S ingle-cell analysis of protein abundance has revealed cell-tocell variation in the form of phenotypic (extrinsic) heterogeneity and intrinsic variation (1). Dynamic processes such as the cell cycle, endocytosis, and meiosis are regulated but also influenced by stochastic events involving small numbers of molecules (e.g., DNA transcription) (2, 3). Similarly, the size and molecular composition of small subcellular organelles are dynamically regulated but still subject to stochastic noise. Studying the biomolecular composition and size of organelles will illuminate the regulation and noise in these dynamically stable systems. A major challenge for such exploration, however, is the development of a combined approach for both resolving small structures below the optical diffraction limit and simultaneously counting biomolecules over several orders of magnitude (Fig. 1A).In the endocytic pathway, both vesicle morphology and biomolecular composition are dynamically regulated along a maturation path. Formation of vesicles, tethering, fusion, and maturation to endosomes are controlled by over 60 proteins in concert with conversion of phosphoinositides (PIs) (4, 5). Phosphatases and PI-kinases produce phosphatidylinositol 3-phosphate (PI3P) from plasma membrane phospholipids (6). PI3P is required for endocytosis (7) and membrane transport to early and late endosomes (8, 9). PI3P regulates the recruitment of proteins (10), such as Rab GTPases, which coordinate many aspects of vesicle identity (11, 12) and maturation to endosomes, including tethe...

Graphical AbstractHighlights d Protein localization and organelle-specific phosphosites upon hepatic LD accumulation d HFD induces contacts between organelles, orchestrating lipid metabolism d LDs sequester compartment-specific proteins upon steatosis d The secretory apparatus redistributes, reducing protein secretion SUMMARY Lipid metabolism is highly compartmentalized between cellular organelles that dynamically adapt their compositions and interactions in response to metabolic challenges. Here, we investigate how diet-induced hepatic lipid accumulation, observed in non-alcoholic fatty liver disease (NAFLD), affects protein localization, organelle organization, and protein phosphorylation in vivo. We develop a mass spectrometric workflow for protein and phosphopeptide correlation profiling to monitor levels and cellular distributions of $6,000 liver proteins and $16,000 phosphopeptides during development of steatosis. Several organelle contact site proteins are targeted to lipid droplets (LDs) in steatotic liver, tethering organelles orchestrating lipid metabolism. Proteins of the secretory pathway dramatically redistribute, including the mis-localization of the COPI complex and sequestration of the Golgi apparatus at LDs. This correlates with reduced hepatic protein secretion. Our systematic in vivo analysis of subcellular rearrangements and organelle-specific phosphorylation reveals how nutrient overload leads to organellar reorganization and cellular dysfunction.

The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single‐molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer.

Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of approximately 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.

We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires widefield illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.