Turn-on mode diarylethenes for bioconjugation and fluorescence microscopy of cellular structures.

The use of photoswitchable fluorescent diarylethenes (fDAEs) as protein labels in fluorescence microscopy and nanoscopy has been limited by labeling inhomogeneity and the need for ultraviolet light for fluorescence activation (on-switching). To overcome these drawbacks, we prepared "turn-on mode" fDAEs featuring thienyl substituents, multiple polar residues, and a reactive maleimide group in the core structure. Conjugates with antibodies and nanobodies displayed complete on-switching and excitation with violet (405 nm) and yellow-green (<565 nm) light, respectively. Besides, they afforded high signal-to-noise ratios and low unspecific labeling in fluorescence imaging. Irradiation with visible light at 532 nm or 561 nm led to transient on-off switching ("blinking") of the fDAEs of double-labeled nanobodies so that nanoscale superresolution images were readily attained through switching and localization of individual fluorophores.

Supramolecular Complex of Cucurbit[7]uril with Diketopyrrolopyrole Dye: Fluorescence Boost, Biolabeling and Optical Microscopy.

New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared; with spectra (H2O, abs. / emission max. 480 / 550 nm; e ~ 19 000, tfl > 4 ns), strong binding with hosts (~560 nM Kd) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595 nm or 655 nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable.

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.

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 Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy.

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit with an alpha-carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live-cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single-digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging.

Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host-guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φfl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70-90 nm optical resolution.

Cleavable Linker Incorporation into a Synthetic Dye-Nanobody-Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy.

A bright and photostable fluorescent dye with a disulfide (S-S) linker and maleimide group (Rho594-S2-mal), as cleavable and reactive sites, was synthesized and conjugated with anti-GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti-GFP NB labeled with one or two Rho594-S2-mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP-NB-dye) were applied in FRET-FLIM assays, confocal imaging, and superresolution STED microscopy.

Bis-Rhodamines Bridged with a Diazoketone Linker: Synthesis, Structure, and Photolysis.

Two fluorophores bound with a short photoreactive bridge are fascinating structures and remained unexplored. To investigate the synthesis and photolysis of such dyes, we linked two rhodamine dyes via a diazoketone bridge (-COCN2-) attached to position 5' or 6' of the pendant phenyl rings. For that, the mixture of 5'- or 6'-bromo derivatives of the parent dye was prepared, transformed into 1,2-diarylacetylenes, hydrated to 1,2-diarylethanones, and converted to diazoketones Ar1COCN2Ar2. The high performance liquid chromatography (HPLC) separation gave four individual regioisomers of Ar1COCN2Ar2. Photolysis of the model compound─C6H5COCN2C6H5─in aqueous acetonitrile at pH 7.3 and under irradiation with 365 nm light provided diphenylacetic acid amide (Wolff rearrangement). However, under the same conditions, Ar1COCN2Ar2 gave mainly α-diketones Ar1COCOAr2. The migration ability of the very bulky dye residues was low, and the Wolff rearrangement did not occur. We observed only moderate fluorescence increase, which may be explained by the insufficient quenching ability of diazoketone bridge (-COCN2-) and its transformation into another (weaker) quencher, 1,2-diarylethane-1,2-dione.

Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels.

The use of photoactivatable dyes in STED microscopy has so far been limited by two-photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB-2SiR, a fluorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water-soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.

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.

Reversibly Photoswitchable Fluorescent Diarylethenes Resistant against Photobleaching in Aqueous Solutions.

Low photostability in aqueous solutions is the main drawback of synthetic photochromic dyes, which limits their switching performance and utility in biology, medicine, and life sciences. Even the most promising photochromes-reversibly photoswitchable diarylethenes (DAEs) with fluorescent "closed" forms-are known to undergo only several tens (typically 20-30) of switching cycles in aqueous solutions. In this work, we introduce water-soluble and highly photostable 1,2-[bis(2-ethyl/2-isobutyl-1-benzothiophene-1,1-dioxide-6-phenyl-3-yl)]perfluorocyclopentenes decorated with four -CONHC(CH2R)3 residues capped with 12 carboxylic acid groups (R = CH2CO2H, O(CH2)2CO2H). Under irradiation with UV (365 nm) and visible light (470 nm), they provide several hundred (for the "rapid" DAEs with isobutyl groups, up to 1000) full switching cycles in aqueous solutions without exclusion of air oxygen (outperforming the photoswitchable and fluorescent protein Dreiklang). The new branching approach based on secondary carboxamides with N-tert-alkyl residues decorated with polar groups may serve as a blueprint for the design of highly fatigue resistant and reversibly photoswitchable fluorescent probes applicable in life sciences as aqueous solutions.

Mono- and bithiophene-substituted diarylethene photoswitches with emissive open or closed forms.

We present a new series of photochromic 1,2-bis(2-ethylbenzo[b]thiophen-3-yl)perfluorocyclopentenes with an oxidized benzothiophene core (O) or a nonoxidized one, decorated with mono- (Th1) and bithiophene (Th2) units attached to positions 6 and 6' (Sy = symmetric) or only to position 6 (As = asymmetric). "Oxidized" compounds have highly fluorescent closed forms emitting in the visible region (yellow to red). The dyes with nonoxidized benzothiophenes possess fluorescent open forms with rather low emission efficiency. The photoswitching kinetics was studied at several wavelengths with UV and visible light. New diarylethenes underwent ring-closure reactions by irradiation with UV light (365 nm, 405 nm), and the reversible ring-opening by irradiation with visible light (470 nm, 530 nm). The on-switching of fluorescence due to the ring-closure reaction was observed also with visible light of 470 nm (to an extent of 10% for compound SyOTh 1 ) and attributed to the Urbach tail effect. Due to a high degree of fluorescence modulation (>270), good fatigue resistance and large fluorescence quantum yield, compound SyOTh 1 emerged as a candidate for single-molecule based super-resolution fluorescence microscopy.

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "Universal Hydrophilizer" or Hydroxyl Groups.

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

Fluorescent Photoswitchable Diarylethenes for Biolabeling and Single-Molecule Localization Microscopies with Optical Superresolution.

A modular assembly of water-soluble diarylethenes (DAEs), applicable as biomarkers for optical nanoscopy, is reported. Reversibly photoswitchable 1,2-bis(2-alkyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes possessing a fluorescent "closed" form were decorated with one or two methoxy group(s) attached to the para-position(s) of phenyl ring(s) and two, four, or eight carboxylic acid groups. Antibody conjugates of these DAEs feature low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and photophysical properties (high emission efficiencies and low cycloreversion quantum yields) enabling their application in superresolution microscopy. Images of tubulin, vimentin, and nuclear pore complexes are presented. The superresolution images can also be acquired by using solely 488 nm light without additional photoactivation with UV light. These DAEs exhibit reversible photoswitching without requiring any additives to the imaging media and open new paths toward the modular design of fluorescent dyes for bioimaging with optical superresolution.

Bichromophoric Compounds with Orthogonally and Parallelly Arranged Chromophores Separated by Rigid Spacers.

Electronic energy transfer (EET) between chromophores is of fundamental importance for many biological processes and optoelectronic devices. However, common models fall short in fully describing the process, especially in bichromophoric model systems with a donor and acceptor connected by a rigid linker providing perpendicular geometries. Herein, we report a novel strategy for preparing bichromophores containing adamantane or 2-(2-adamantylidene)adamantane as rigid spacers, providing a fixed distance between chromophores, and their parallel or perpendicular arrangement without chromophore rotation. New fluorophores were developed and linked via spiroatoms. Bichromophores with identical (blue-blue) or different (blue-red) chromophores were synthesized, either in orthogonal or parallel geometry. These were characterized by absorption/fluorescence spectroscopy, time-resolved fluorescence anisotropy, and fluorescence antibunching measurements. Based on the Förster point-dipole approximation, EET efficiencies were estimated by using geometrical parameters from (time-dependent) density functional calculations. For bichromophores with parallel geometry, the predicted EET efficiencies were near unity and fit the measurements. In spite of estimated values around 0.4 and 0.5, 100 % efficiency was observed also for bichromophores with orthogonal geometry. The new rigid scaffolds presented here open new possibilities for the synthesis of bichormophores with well-defined parallel or perpendicular geometry.

"Reduced" Coumarin Dyes with an O-Phosphorylated 2,2-Dimethyl-4-(hydroxymethyl)-1,2,3,4-tetrahydroquinoline Fragment: Synthesis, Spectra, and STED Microscopy.

Large Stokes-shift coumarin dyes with an O-phosphorylated 4-(hydroxymethyl)-2,2-dimethyl-1,2,3,4-tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N-substituted and O-protected 1,2-dihydro-7-hydroxy-2,2,4-trimethylquinoline was oxidized with SeO2 to the corresponding α,ß-unsaturated aldehyde and then reduced with NaBH4 in a "one-pot" fashion to yield N-substituted and 7-O-protected 4-(hydroxymethyl)-7-hydroxy-2,2-dimethyl-1,2,3,4-tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes ("reduced coumarins") and 1,2-dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The "reduced coumarins" were found to be more photoresistant and brighter than their 1,2-dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N-hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red-emitting coumarin dye bearing a betaine fragment at the C-3-position showed excellent performance in stimulation emission depletion (STED) microscopy.

Palladium Nanoparticles Embedded in a Layer-by-Layer Nanoreactor Built with Poly(Acrylic Acid) Using "Electro-Click Chemistry".

Palladium nanoparticles (Pd NPs) were formed by electrochemical reduction of Pd(NH3)4(3+) 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 Cu(2+) 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.

Förster resonance energy transfer mediated enhancement of the fluorescence lifetime of organic fluorophores to the millisecond range by coupling to Mn-doped CdS/ZnS quantum dots.

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.

Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy.

Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent "closed" forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology-related super-resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.