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We report synthesis and photophysical studies of spirorhodamines (SRA) with amide N-aliphatic substituents, including measurements of single-crystal X-ray structures, fluorescence emission, and quantum mechanical calculations. Variations in the equilibrium position between closed and open states, a transition that is catalysed under acidic conditions, of several SRAs sharing the same central chromophore (Rhodamine 6G) were measured. For the different substituents in the lactam position, the pH-dependent lactam ring opening reaction displays a strong correlation with the volume of the functional group. The open form shows similar absorption and emission spectrum, fluorescence efficiency and emission lifetime, regardless of the nature of the substituents. The molecular volume of the substituent is a simple and straightforward molecular design feature to control the equilibrium between the dark and bright states of the probe, without any further changes in the emission properties of the fluorescent form. These results allow rational design of pH-sensitive aliphatic SRAs fluorescent probes. Fil: Di Paolo, Matias Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Alday, Julieta Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Michel Torino, Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Suarez, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

Sequential adsorption of PdCl42- within weak polyelectrolyte layer-by-layer (LbL) self-assembled multilayer films with further electrochemical reduction to yield Pd0 nanoparticles (Pd-NPs) has been demonstrated. The electrocatalytic hydrogenation (ECH) of model molecules such as acetophenone and benzophenone on Pd-NPs of different sizes (6 to 35 nm) and bulk Pd crystal surface in hydroalcoholic acid solution has been investigated. Distribution of reaction products (secondary alcohols and alkanes) and faradaic yield was systematically investigated. While the polyelectrolyte multilayers act as nanoreactors by confining PdCl42- ions and preventing the formation of large crystals, their presence also alters the hydrogenation reaction and therefore heat treated surfaces showed only the effect of nanocrystal size on the reaction selectivity and faradaic yield. This journal is Fil: Villalba, Matias Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Calvo, Ernesto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as “hidden” markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ∼750 nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330–350 nm (molar extinction coefficient (ε)≈104 M−1 cm−1) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511–633 and 525–653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε= 3–8×104 M−1 cm−1 and fluorescence quantum yields (ϕ)=40–85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375–420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3–6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20–38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10–40 % of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a “dark” non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified. Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Mitronova, Gyuzel Yu.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Boyarskiy, Vadim P.. St. Petersburg State University; Rusia Fil: Hebisch, Elke. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Geisler, Claudia. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Wurm, Christian A.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Willig, Katrin I.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry ; Alemania

Nanoparticle-based temperature imaging is an emerging field of advanced applications. Herein, the sensitivity of the phosphorescence of tris(bipyridine)ruthenium(II)-doped silica nanoparticles towards temperature is studied. 130 nm size particles were prepared by a modification of Stöber’s method, that allows the incorporation of Ru[(bpy)3]2+ into the outer particle shell. The entrapped Ru[(bpy)3]2+ retains its photophysical properties, yet the emission of the particles is not affected by the presence of O2, neither by anionic quenchers; quenching by MV2+, on the other hand, is strongly dependent on pH. Between 20 and 60 °C, the steady-state emission of the particles decreases linearly with increasing temperature. The slope of the straight line diminishes slightly on thermal cycling, but soon stabilizes. Fluorescence measurements by scanning confocal microscopy indicate that the silica nanoparticles doped with Ru[(bpy)3]2+ can indeed be employed to probe thermal processes in micro-environments. Fil: Mirenda, Martin. Comision Nacional de Energia Atomica. Centro Atomico Constituyentes; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Levi, Valeria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bossi, Mariano Luis. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bruno, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bordoni, Andrea Veronica. Comision Nacional de Energia Atomica. Centro Atomico Constituyentes; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Regazzoni, Alberto Ernesto. Comision Nacional de Energia Atomica. Centro Atomico Constituyentes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Wolosiuk, Alejandro. Comision Nacional de Energia Atomica. Centro Atomico Constituyentes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The synthesis, characterization, structural analysis and fluorescence properties of two rhodamine 6G derivatives are described, namely a propargylamine derivative, 3′,6′-bis(ethylamino)-2′,7′-dimethyl-2-(methylcyanide)spiro[isoindole-1,9′-xanthen]-3(2H)-one (I), and a γ-aminobutyric acid (GABA) derivative, 3′,6′-bis(ethylamino)-2′,7′-dimethyl-3-oxospiro[isoindole-1,9′-xanthen]-2(3H)-yl)butyricacid (II). Both structures are compared with four similar ones from the Cambridge Structural Database (CSD), and the interactions involved in the stabilization are analyzed using the atoms in molecules (AIM) theory. Finally, a single-crystal in-situ reaction study is presented, carried out by fluorescence methods, which enabled the 'opening' of the spirolactam ring in the solid phase. Fil: Di Paolo, Matias Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Baggio, Ricardo Fortunato. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina Fil: Suarez, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina

During the last decade far-field fluorescence microscopy methodshave evolved that have resolution far below the wavelength oflight. To outperform the limiting role of diffraction, all thesemethods, in one way or another, switch the ability of a moleculeto emit fluorescence. Here we present a novel rhodamine amidethat can be photoswitched from a nonfluorescent to a fluorescentstate by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precisiondown to 15 nm in the focal plane. Two-photon induced nonlinearphotoswitching of this marker with continuous-wave illuminationoffers optical sectioning with simple laser equipment.Future synthesis of similar compounds holds great promise forcost-effective fluorescence nanoscopy with noninvasive optical sectioning. Fil: Fölling, Jonas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Riedel, Jennifer Denise. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Schönle, Andreas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Egner, Alexander. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Eggeling, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

We introduce far-field fluorescence nanoscopy with ordinaryfluorophores based on switching the majority of them to ametastable dark state, such as the triplet, and calculating theposition of those left or those that spontaneously returned to theground state. Continuous widefield illumination by a single laserand a continuously operating camera yielded dual-color imagesof rhodamine- and fluorescent protein–labeled (living) samples,proving a simple yet powerful super-resolution approach. Fil: Fölling, Jonas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bock, Hannes. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Wurm, Christian A. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hein, Birka. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Jakobs, Stefan. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Eggeling, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W. Max Planck Institute for Biophysical Chemistry; Alemania

Palladium nanoparticles (Pd NPs) were formed by electrochemical reduction of Pd(NH3)43+ ions entrapped by ion exchange in poly(acrylic acid) (PAA) multilayer films grown by the Sharpless "click reaction." The alkyne (PAAalk) and azide (PAAaz) groups were covalently bound to the PAA, and the catalyzed buildup of the multilayer film was performed by electrochemical reduction of Cu2+ to Cu+. The size of the Pd NPs formed in Au/(PAAalk)3(PAAaz)2 multilayer films by the click reaction, that is, 50 nm, is larger than that of similar Pd NPs formed in electrostatically bound Au/(PAA)3(PAH)2 nanoreactors, that is, 6-9 nm, under similar conditions. A combination of electrochemical methods and electrochemical quartz crystal microbalance, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), ellipsometry, and scanning electron microscopy has been used to follow these processes. Cyclic voltammetry of the resulting Pd NPs in a 0.1 M H2SO4 solution at 0.1 V·s-1 shows the PdO reduction peak at the same potential as that on the clean Pd surface unlike the NPs formed in electrostatically self-assembled Au/(PAA)3(PAH)2 nanoreactors with a 0.2 V shift in the cathodic direction most probably because of the strong adsorption of amino groups on the Pd NP surfaces. Fil: Villalba, Matias Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: del Pozo, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Calvo, Ernesto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

A coumarin fluorophore and an oxazine photochrome can be integrated within the same molecular skeleton and connected covalently to a secondary antibody. Illumination of the antibody-dyad conjugate at an appropriate activation wavelength opens the oxazine ring reversibly and shifts bathochromically the ground-state absorption of the coumarin component. Selective excitation of the photochemical product then produces significant fluorescence and allows the detection of activated bioconjugates at the single-molecule level. Such fluorescence activation events can be exploited to resolve temporally individual emitters and reconstruct images of immunolabeled cells with subdiffraction resolution. Relying on a similar conjugation protocol, a model compound, incorporating the same chromophore of the photochemical product, can also be connected covalently to a secondary antibody. Stimulated emission can be exploited to deplete the excited state of the bioconjugated chromophores and switch their fluorescence off. These operating principles for fluorescence switching also permit the imaging of immunolabeled cells with subdiffraction resolution. Thus, these photoswitchable molecules, in combination with the labeling ability of antibodies, can evolve into valuable probes for bioimaging with superresolution. Fil: Cusido, Janet. University of Miami; Estados Unidos Fil: Ragab, Sherif Shaban. University of Miami; Estados Unidos. National Research Centre; Estados Unidos Fil: Thapaliya, Ek Raj. University of Miami; Estados Unidos Fil: Swaminathan, Subramani. University of Miami; Estados Unidos Fil: Garcia-Amorós, Jaume. University of Miami; Estados Unidos Fil: Roberti, Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Araoz, Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Mazza, Mercedes M. A.. University of Miami; Estados Unidos Fil: Yamazaki, Shiori. University of Miami; Estados Unidos Fil: Scott, Amy M.. University of Miami; Estados Unidos Fil: Raymo, Françisco M.. University of Miami; Estados Unidos Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

We designed a strategy to activate fluorescence under the influence of optical stimulations based on the intermolecular transfer of protons. Specifically, the illumination of a 2-nitrobenzyl derivative at an activating wavelength is accompanied by the release of hydrogen bromide. In turn, the photogenerated acid encourages the opening of an oxazine ring embedded within a halochromic compound. This structural transformation extends the conjugation of an adjacent coumarin fluorophore and enables its absorption at an appropriate excitation wavelength. Indeed, this bimolecular system offers the opportunity to activate fluorescence in liquid solutions, within rigid matrixes and inside micellar assemblies, relying on the interplay of activating and exciting beams. Furthermore, this strategy permits the permanent imprinting of fluorescent patterns on polymer films, the monitoring of proton diffusion within such materials in real time on a millisecond time scale, and the acquisition of images with spatial resolution at the nanometer level. Thus, our operating principles for fluorescence activation can eventually lead to the development of valuable photoswitchable probes for imaging applications and versatile mechanisms for the investigation of proton transport. © 2012 American Chemical Society. Fil: Swaminathan, Subramani. University of Miami; Estados Unidos Fil: Petriella, Marco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Deniz, Erhan. University of Miami; Estados Unidos Fil: Cusido, Janet. University of Miami; Estados Unidos Fil: Baker, James D.. University of Miami; Estados Unidos Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Raymo, Françisco M.. University of Miami; Estados Unidos

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group. Fil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Wurm, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Sednev, Maksim V.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

Manganese-doped CdS/ZnS quantum dots have been used as energy donors in a Förster-like resonance energy transfer (FRET) process to enhance the effective lifetime of organic fluorophores. It was possible to tune the effective lifetime of the fluorophores by about six orders of magnitude from the nanosecond (ns) up to the millisecond (ms) region. Undoped and Mn-doped CdS/ZnS quantum dots functionalized with different dye molecules were selected as a model system for investigating the multiple energy transfer process and the specific interaction between Mn ions and the attached dye molecules. While the lifetime of the free dye molecules was about 5 ns, their linking to undoped CdS/ZnS quantum dots led to a long effective lifetime of about 150 ns, following a non-exponential transient. Manganese-doped core-shell quantum dots further enhanced the long-lasting decay time of the dye to several ms. This opens up a pathway to analyse different fluorophores in the time domain with equal spectral emissions. Such lifetime multiplexing would be an interesting alternative to the commonly used spectral multiplexing in fluorescence detection schemes. Fil: Kaiser, Uwe. Philipps-University Marburg, ; Alemania Fil: Sabir, Nadeem. Philipps-University Marburg, ; Alemania Fil: Carrillo-Carrion, Carolina. Philipps-University Marburg, ; Alemania Fil: Del Pino, Pablo. CIC biomaGUNE; España Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Heimbrodt, Wolfram. Philipps-University Marburg, ; Alemania Fil: Parak, Wolfgang J.. Philipps-University Marburg, ; Alemania. CIC biomaGUNE; España

New photostable rhodamine dyes represented by the compounds 1a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N′,N-bis(2,2,2-trifluoroethyl) derivatives 1e, 1i, 1j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4′ and 5′. Two fluorine atoms were introduced into the positions 2′ and 7′ of the 3′,6′-diaminoxanthene fragment in compounds 1a-d, 1i-l and 1m-r. The new rhodamine dyes may be excited with λ= 488 or 514 nm light; most of them emit light at λ = 512-554 nm (compounds 1q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98%), relatively long excited-state lifetimes (>3ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30% SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH 2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Fil: Mitronova, Gyuzel Yu. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Wurm, Christian A.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Meyer, Lars. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Moneron, Gael. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bretschneider, Stefan. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Eggeling, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Jakobs, Stefan. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

Samples of a nematic mixture of ZLI1132 and of a twisted nematic mixture composed of ZLI1132 and chiral inductor S811, including 1%-10% (w/w) 4-N,N-dimethylaminoazobenzene (DAB), (4′-nitro)-4-N,Ndimethylaminoazobenzene (NDAB), spiropyran (SP), or spirooxazine (SO) were irradiated to produce the photochromic transformation of the dopant. The changes in the system were monitored by time-resolved transmission spectroscopy, time-resolved birefringence, or polarized Raman scattering. The medium sensitivity of the kinetics and spectroscopy of some of the probes was used to derive information on polarity of the medium. In the systems studied, apart from the changes in absorption spectrum, great changes in birefringence can be photoinduced and the order of the nematic phase can be changed in either direction, depending on the dopant. The open form of SP can discriminate orientation polarity. Although the polarity parallel to the mesogenic director is similar to that for acetone, the perpendicular orientation has a polarity similar to acetonitrile. In agreement with this observation, the kinetics of the Z f E isomerization of NDAB, oriented parallel to the mesogenic director, also experiences a polarity similar to that for acetone. The decay rate constant of the open form of SP displays a linear relationship between its Arrhenius parameters, which is universal in a great variety of homogeneous solvents, solvent mixtures, and liquid crystals, therefore validating the hypothesis that the same type of transformation is observed in all these cases, namely, the decay of the open form monomer. The dopants used have been proven to be adequate probes of bulklike properties in locally heterogeneous systems as liquid crystals. Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Murgida, Daniel Horacio. Universität zu Berlin; Alemania Fil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina

We synthesized five fluorophore-photochrome dyads designed to switch reversibly between nonfluorescent and fluorescent isomers under optical control. These compounds pair an oxazine photochrome to a biphenyl, fluorene, pyrene, coumarin, or cyanine fluorophore in their molecular skeleton and can be prepared in a single step from known precursors in yields ranging from 30 to 63%. Nuclear magnetic resonance spectroscopy indicates that the oxazine ring of these compounds opens and closes spontaneously on a millisecond time scale in acetonitrile at ambient temperature. Under these conditions, the fraction of ring-open isomer at equilibrium is negligible in all instances with the exception of the cyanine derivative, which instead is almost exclusively in this form. Absorption and emission spectroscopies demonstrate, however, that the fraction of ring-open isomer is sensitive to solvent polarity and increases with a transition from acetonitrile to methanol. Alternatively, the ring-open isomer can be populated photochemically or trapped with the addition of acid. In both instances, the characteristic absorption and emission bands of the 3H-indolium chromophores, embedded within the ring-open species, can clearly be observed in the visible region. In the case of the coumarin derivative, the brightness of this chromophoric fragment is sufficiently high to permit the imaging of individual molecules with excellent signal-to-noise ratios. In fact, the fluorescence of single fluorophore-photochrome dyads can be activated under the influence of ultraviolet inputs and the resulting species can be localized with nanoscale precision under visible illumination. Indeed, subdiffraction images of polymer nanoparticles, doped with this particular dyad, can be reconstructed with nanoscale resolution. Thus, our operating principles for fluorescence switching at the single-molecule level can offer the opportunity to overcome diffraction and, eventually, lead to the development of an entire family of probes for super-resolution fluorescence imaging. © 2012 American Chemical Society. Fil: Deniz, Erhan. University of Miami; Estados Unidos Fil: Tomasulo, Massimiliano. University of Miami; Estados Unidos Fil: Cusido, Janet. University of Miami; Estados Unidos Fil: Yildiz, Ibrahim. University of Miami; Estados Unidos Fil: Petriella, Marco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Sortino, Salvatore. University of Catania; Italia Fil: Raymo, Françisco M.. University of Miami; Estados Unidos

We review macroscopic properties of materials that can be modulated by light through switching between the two stable states of a photochromic system. In special cases, the photochromic compound is the only component of the material, but in most cases it is either embedded or covalently linked to a suitable host, which is normally a liquid crystalline phase, a glassy or a rubbery polymer, or an interface. We analyze examples that illustrate changes in refractive index, transmission of polarized light, reflectivity, light dispersion, polarization, charge transport capability, dielectrical properties, stable phase, microscopic surface relief in large areas, hydrophobicity of the surface, permeability, and even bulk mechanical deformations. The basis of such changes at a molecular level is associated with a difference in the shape, dipole moment, polarizability, or electronic features of the photochromic species, and their different interaction with the environment. In some cases, studies were performed at the single molecule level. Each effect is discussed stressing the favorable features of the particular photochromic compounds to induce the changes and its interaction with the environment that make it suitable for the appointed application. © 2011 Elsevier B.V. Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina

Photochromic compounds with improved performance in pure water have been prepared and characterized. They possess an asymmetrical and extended conjugation system with a 1,2-bis(3-thienyl)perfluorocyclopentene core, additional thiophene rings connected with sulfonic acid residues, as well as terminal pyrid-4-yl and 4-alkoxyphenyl groups. A multistep synthetic route to a key compound with an amino group required for further derivatization has been developed. The complete photocyclization reaction of the initial "open ring" compound (with absorption maximum at 340 nm in MeOH or water) can be performed with a diode laser in pure water or aqueous buffer solutions (without added or-ganic solvents) under irradiation at 366-375 nm, and the reverse ring-opening reaction of the colored "closed ring" compound (λmax = 628 or 624 nm in MeOH or water) takes place under irradiation with visible green or red light (>500 nm). Building block 57, with a secondary amino group, can further be used in the synthesis of practically important, reversibly switchable, fluorescent compounds in which the fluorescent dye (donor) is attached to a photochromic unit (acceptor), and the resonant energy transfer from a donor to the colored form of an acceptor provides a reversible quenching of the fluorescence signal in aqueous solutions. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Fil: Polyakova, Svetlana M.. Max Planck Institute for Biophysical Chemistry; Alemania. Institute of Organic Chemistry and Biochemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

The design, synthesis, and evaluation of new rhodamine spiroamides are described. These molecules have applications in optical nanoscopy based on random switching of the fluorescent single molecules. The new markers may be used in (co)localization studies of various objects and their (mutual) positions and shape can be determined with a precision of a few tens of nanometers. Multicolor staining, good photoactivation, a large number of emitted photons, and selective chemical binding with amino or thiol groups were achieved due to the presence of various functional groups on the rhodamine spiroamides. Rigidized sulfonated xanthene fragment fused with six-membered rings, N,N′-bis(2,2,2-trifluoroethyl) groups, and a combination of additional double bonds and sulfonic acid groups with simple aliphatic spiroamide residue provide multicolor properties and improve performance of the rhodamine spiroamides in photoactivation and bioconjugation reactions. Having both essential parts of the photoswitchable assembly - the switching and the fluorescent (reporter) groups - combined in one chemical entity make this approach attractive for further development. A series of rhodamine spiroamides is presented along with characterizations of their most relevant properties for application as fluorescent probes in single-molecule switching and localization microscopy. Optical images with resolutions on the nanometer scale illustrate the potential of the labels in the colocalization of biological objects and the two-photon activation technique with optical sectioning. Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fölling, Jonas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Boyarskiy, Vadim P.. Max Planck Institute for Biophysical Chemistry; Alemania. St. Petersburg State University; Rusia Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

We combine far-field fluorescence nanoscopy through serializedrecording of switchable emitters with polarization-sensitive fluorescencedetection. In addition to imaging with nanoscale spatial resolution, thistechnique allows determination of the fluorescence anisotropy of eachdetected dipole emitter and thus an estimate of its rotational mobility. Subpopulationsof fluorescent markers can thus be separated based on theirinteraction with the sample. We applied this new functional nanoscopy toimaging of living mammalian cells. Fil: Testa, Ilaria. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Schönle, Andreas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: V. Middendorff, Claas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Geisler, Claudia. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Wurm, Christian A.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Stiel, Andre C.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Jakobs, Stefan. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Eggeling, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Egner, Alexander. Max Planck Institute for Biophysical Chemistry; Alemania

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques. Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Folling, Jonas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Boyarskiy, Vadim P.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Egner, Alexander. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Eggeling, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Schonle, Andreas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

New fluorescent photochromic compounds (1-H and 1-Boc)have been synthesized and characterized in different solvents.The fluorescence emission can be switched “on” and“off” with visible light and UV, respectively, by means of thephotochromic reaction. The emission wavelength and efficiencystrongly depend on the polarity of the solvent. Thecompounds show a positive solvatochromic effect in theemission maxima, and their fluorescence quantum yield decreasesas the solvent’s polarity increases (from cyclohexaneto dioxane). In solvents more polar than dioxane the emissionis too weak and therefore undetectable, and thus 1-H and 1-Boc behave as “normal” photochromic compounds. The photochromic reaction is also sensitive to the environment. A decreaseof more than an order of magnitude was found for thequantum yield of the colouring reaction (ΦOFCF) for 1-H inethanol compared with cyclohexane, and an about threefolddecrease in ΦOFCF was observed for the compound 1-Bocin polar solvents (compared with apolar solvents). For bothcompounds the ring-opening reaction was found not to dependenton the solvent. The novel fluorescent molecularswitches 1-H and 1-Boc are able to probe the polarity of theirmicroenvironment. Fil: Yan, Sergey F.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

We have designed and synthesized a new functional (amino reactive)highly efficient fluorescent molecular switch (FMS) with a photochromicdiarylethene and a rhodamine fluorescent dye. The reactive group in thisFMS -N-hydroxysuccinimide ester- allows selective labeling of aminocontaining molecules or other materials. In ethanolic solutions, thecompound displays a large fluorescent quantum yield of 52% and alarge fluorescence modulation ratio (94%) between two states that maybe interconverted withred and near-UV light. Silica nanoparticles incorporatingthe new FMS were prepared and characterized, and their spectroscopicand switching properties were also studied. The dye retained itsproperties after the incorporation into the silica, thereby allowing lightinducedreversible high modulation of the fluorescence signal of a singleparticle for up to 60 cycles, before undergoing irreversible photobleaching.Some applications of these particles in fluorescence microscopy arealso demonstrated. In particular, subdiffraction images of nanoparticleswere obtained, in the focal plane of a confocal microscope. Fil: Fölling, Jonas. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Polyakova, Svetlana. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Van Blaaderen, Alfons. Utrecht University; Países Bajos Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Hell, Stefan W.. Utrecht University; Países Bajos

Highly water soluble fluorescentdyes were synthesized and transformedinto new amino reactive fluorescentlabels for biological microscopy.To this end,rhodamine 8 (preparedfrom 7-hydroxy-1,2,3,4-tetrahydroquinoline(7) and phthalic anhydride in85% aq. H3PO4) was sulfonated with30% SO3 in H2SO4 and afforded thewater soluble disulfonic acid 3a (64%).Amidation of the carboxy group in 3awith 2-(methylamino)ethanol in thepresence of O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium·PF6(HATU) led to alcohol 3b (66%),which was transformed into the aminoreactive mixed carbonate 3d with di(Nsuccinimidyl)carbonate and Et3N. Reactionof the carboxy group in 3a withMeNHACHTUNGTRENUNG(CH2)2CO2Me and N,N,N’,N’-tetramethyl-O-(N-succinimidyl)-uronium·BF4 (TSTU) yielded methylester 13. After saponification of the aliphaticcarboxy group in 13,the compoundwas converted into NHS-ester3e (using HATU and Et3N). Heatingof 7 with trimellitic anhydride inH3PO4 gave a mixture of dicarboxylicacids 14 and 15 (1:1). Regioisomer 15was isolated,sulfonat ed with 30% SO3in H2SO4,and disulfonic acid 3 f wasused for the synthesis of the monoNHS-ester 3g,in which the stericallyunhindered carboxy group was selectivelyactivated (with N-hydroxysuccinimide,HA TU,and Et3N). The sulfonatedrhodamines 3b, c and f are solublein water (up to 0.1m),have excellentphotostabilities and large fluorescencequantum yields. Subdiffractionresolution images of tubulin filamentsof mammalian cells stained with thesedyes illustrate their applicability aslabels for stimulated emission depletionmicroscopy and other fluorescencetechniques. Fil: Boyarskiy, Vadim P.. Max Planck Institute for Biophysical Chemistry; Alemania. Saint Petersburg State University; Rusia Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hein, Birka. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania