Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561 nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560 , PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561 nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561 nm MINFLUX imaging.

Synthesis of Thioxanthone 10,10-Dioxides and Sulfone-Fluoresceins via Pd-Catalyzed Sulfonylative Homocoupling.

Our report describes the facile and scalable preparation of 9H-thioxanthen-9-one 10,10-dioxides via Pd-catalyzed sulfonylative homocoupling of the appropriately substituted benzophenones. This transformation provides a straightforward route to previously unreported sulfone-fluoresceins and -fluorones. Several examples of these red fluorescent dyes have been prepared, characterized, and evaluated as live-cell permeant labels compatible with super-resolution fluorescence microscopy with 775 nm stimulated emission depletion.

Bioorthogonal Caging-Group-Free Photoactivatable Probes for Minimal-Linkage-Error Nanoscopy.

Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the photoactivatable xanthone (PaX) core with a tetrazine group, we achieve minimally sized and highly sensitive molecular dyads for the selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit the excited state quenching properties of the tetrazine group to attenuate the photoactivation rates of the PaX, and further reduce the overall fluorescence emission of the photogenerated fluorophore, providing two mechanisms of selectivity to reduce the off-target signal. Coupled with MINFLUX nanoscopy, we employ our dyads in the minimal-linkage-error imaging of vimentin filaments, demonstrating molecular-scale precision in fluorophore positioning.

Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores.

The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized for a high photon yield and rather long on-times (tens of milliseconds). Here, accelerated MINFLUX nanoscopy with up to a 30-fold gain in localization speed is presented. The improvement is attained by designing spontaneously blinking fluorescent markers with remarkably fast on-times, down to 1-3 ms, matching the iterative localization process used in a MINFLUX microscope. This design utilizes a silicon rhodamine amide core, shifting the spirocyclization equilibrium toward an uncharged closed form at physiological conditions and imparting intact live cell permeability, modified with a fused (benzo)thiophene spirolactam fragment. The best candidate for MINFLUX microscopy (also suitable for STORM imaging) is selected through detailed characterization of the blinking behavior of single fluorophores, bound to different protein tags. Finally, optimization of the localization routines, customized to the fast blinking times, renders a significant speed improvement on a commercial MINFLUX microscope.

Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy.

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

N-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins.

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their O-unprotected forms. Herein, we report rationally designed N-cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from N-cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells.

A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy.

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging super-resolution methods. Photoactivatable dyes offer substantial improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group-free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogues, yielding a family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell labelling for conventional microscopy, as well as the coordinate-stochastic and deterministic nanoscopies STED, PALM and MINFLUX.

Photoactivatable Fluorescent Dyes with Hydrophilic Caging Groups and Their Use in Multicolor Nanoscopy.

We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity (visible or infrared) light of about twice the wavelength. The caged fluorescent dyes and labels derived therefrom display high water solubility and convert upon photoactivation into validated super-resolution and live-cell-compatible fluorophores. In combination with popular fluorescent markers, multiple (up to six)-color images can be obtained with stimulated emission depletion nanoscopy. Moreover, individual fluorophores can be localized with precision <3 nm (standard deviation) using MINSTED and MINFLUX techniques.

MINSTED fluorescence localization and nanoscopy.

We introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1,000 detections per fluorophore provide a localization precision of 1-3nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a -100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

Modular Synthetic Approach to Silicon-Rhodamine Homologues and Analogues via Bis-aryllanthanum Reagents.

A modular synthetic approach toward diverse analogues of the far-red fluorophore silicon-rhodamine (SiR), based on a regioselective double nucleophilic addition of aryllanthanum reagents to esters, anhydrides, and lactones, is proposed. The reaction has improved functional group tolerance and represents a unified strategy toward cell-permeant, spontaneously blinking, and photoactivatable SiR fluorescent labels. In tandem with Pd-catalyzed hydroxy- or aminocarbonylation, it serves a streamlined synthetic pathway to a series of validated live-cell-compatible fluorescent dyes.

Triarylmethane Fluorophores Resistant to Oxidative Photobluing.

Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.

Fluorescent dyes and probes for super-resolution microscopy of microtubules and tracheoles in living cells and tissues.

We introduce fluorogenic tubulin probes based on the recently reported fluorescent dyes (510R, 580CP, GeR and SiR) and chemotherapy agents - taxanes (docetaxel, cabazitaxel and larotaxel). The cytotoxicity of the final probe, its staining performance and specificity strongly depend on both components. We found correlation between the aggregation efficiency (related to the spirolactonization of fluorophore) and cytotoxicity. Probe optimization allowed us to reach 29 ± 11 nm resolution in stimulated emission depletion (STED) microscopy images of the microtubule network in living human fibroblasts. Application to living fruit fly (Drosophila melanogaster) tissues highlighted two distinct structures: microtubules and tracheoles. We identified 6-carboxy isomers of 580CP and SiR dyes as markers for chitin-containing taenidia, a component of tracheoles. STED microscopy revealed correlation between the taenidia periodicity and the diameter of the tracheole. Combined tubulin and taenidia STED imaging showed close interaction between the microtubules and respiratory networks in living tissues of the insect larvae.

PONy Dyes: Direct Addition of P(III) Nucleophiles to Organic Fluorophores.

Nucleophilic addition of phosphinic acid, phosphites, sodium dialkyl phosphites, phosphoramidites, phosphinites, and phosphonites to highly polarized or cationic fluorophores, followed by oxidation, results in new "PONy" dyes with auxochromic phosphinate, phosphonate, or phosphonamidate groups. The reaction was applied to a wide variety of coumarins, (thio)pyronins, and N-alkylacridinium and 5,6-dihydrobenzo[c]xanthen-12-ium salts as well as a meso-chlorinated BODIPY to provide compact dyes with red-shifted absorption and emission bands and Stokes shifts up to 8200 cm-1.

Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins.

A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resolution imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.

High-Affinity Functional Fluorescent Ligands for Human ß-Adrenoceptors.

Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting ß-adrenergic receptor (ßAR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates nanomolar binding affinity and up to nine-fold ß2AR selectivity over ß1AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of ß2ARs stained at the native expression level on pancreatic CAPAN cells provides two-fold improvement in lateral optical resolution over confocal mode and reveals the formation of receptor microdomains. These probes retain their functional (agonist or antagonist) properties, allowing simultaneous modulation of cyclic adenosine monophosphate (cAMP) levels and receptor internalization as well as imaging receptor localization.

Cell-Permeant Large Stokes Shift Dyes for Transfection-Free Multicolor Nanoscopy.

We designed cell-permeant red-emitting fluorescent dye labels with >140 nm Stokes shifts based on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-imino-10-germaxanthone fluorophores. The corresponding probes selectively targeting mitochondria, lysosomes, and F-actin demonstrate low toxicity and enable stimulated emission depletion (STED) nanoscopy in neurons, human fibroblasts, U2OS, and HeLa cells. In combination with known small Stokes shift dyes, our probes allow live-cell three-color STED nanoscopy of endogenous targets on popular setups with 775 nm STED wavelength.

Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED.

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection.

The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 µm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.