Photoactivatable Dyes Research Articles

Optimized Properties and Synthesis of Photoactivatable Diazoketorhodamines Facilitate and Enhance High-Throughput Single-Molecule Tracking.

Photoactivatable (PA) rhodamine dyes are widely used in single-molecule tracking (SMT) and a variety of other fluorescence-based imaging modalities. One of the most commonly employed scaffolds uses a diazoketone to lock the rhodamine in the nonfluorescent closed form, which can be activated with 405 nm light. However, poor properties of previously reported dyes require significant washing, which can be resource- and cost-intensive, especially when performing microscopy in a large scale and high-throughput fashion. Here, we report improved diazoketorhodamines that perform exceptionally well in single-molecule tracking microscopy. We also report on the optimization of an improved synthetic method for further iteration and tailoring of diazoketorhodamines to the requirements of a specific user.

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.

Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy

AbstractNew 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.

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.

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.

Photodynamic treatment affects the secreted antioxidant and glycoside hydrolases activities produced by Humicola grisea var. thermoidea and Penicillium echinulatum in agro-industrial substrates

The exposure of fungi to photoactivatable dyes can modify the expression of enzymes of interest in several areas such as industrial enzyme production, bioremediation of urban residues and nanocomposites stabilization. Alternatives that favor the use of plant biomass, or improve its exploitation should be prioritized, considering the scarcity of fossil resources and their polluting nature. Here, we investigated the effect of photodynamic treatment (PDT) mediated by an aluminum-phthalocyanine chloride nanoemulsion (AlPc-NE) in two hydrolases-producing filamentous fungi, Humicola grisea and Penicillium echinulatum, and its implication in the hydrolysis of agro-industrial substrates as steam exploded sugarcane-bagasse (SCB) and wheat bran (WB). The fungi hyphae were exposed to different concentrations of AlPc-NE and photosensitizer internalization was measured by spectrophotometry and fluorescence microscopy. Once the photosensitizer retention patterns were established, PDT mediated by 300 and 600 nM AlPc-NE was performed and the cultures viability was analyzed. After PDT, H. grisea and P. echinulatum were grown in SCB, WB or glucose and subsequently analyzed after 96 h. The amount of soluble secreted proteins, total antioxidant activity in the supernatant, CMCase, xylanase and FPase enzymes activity were evaluated. After irradiation, there was no change in the cultures growth or viability. Changes in the total amount of secreted protein, total antioxidant activity, CMCase and xylanase activities were observed after PDT in a photosensitizer concentration-dependent manner, and accordingly to the evaluated substrate and fungus species. Xylanase activity was the most impacted while FPase remained virtually unchanged in both fungi species. We concluded that PDT can be employed to modulate the production of certain fungal hydrolases of industrial interest.

Introduction of the Functional Amino Group at the meso Position of Cy3 and Cy5 Dyes: Synthesis, Stability, Spectra and Photolysis of 4‐Amino‐1‐diazo‐2‐butanone Derivatives**

AbstractSearching for photoconvertible or photoactivatable dyes, we considered trimethine (Cy3) and pentamethine (Cy5) fluorophores with a 4‐amino‐1‐diazo‐2‐butanone fragment (H2N(CH2)2COCH=N2) attached to the meso (γ) position of the polymethine chain via the amino group. The Cy3 derivative was prepared. All Cy5 derivatives with the basic amino group at this position decomposed with breaking the polymethine chain. However, amino‐squaraine based Cy5 dyes were found to be stable and accessible. Photolysis of Cy3 and squaraine‐based Cy5 dyes with a 4‐amino‐1‐diazo‐2‐butanone fragment was accompanied with Wolff rearrangement and led to pyrrolidones (in methanol and aprotic solvents) formed via intramolecular cyclization of the intermediate ketenes. In aqueous acetonitrile, ketenes reacted with water and gave carboxylic acids. Both products were non‐fluorescent, though the model Cy5 dyes (without the squaraine fragment) with acylated amino and N‐methylamino groups were found to be fluorescent.

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.

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.

A Molecular Logic Gate Enables Single-Molecule Imaging and Tracking of Lipids in Intracellular Domains.

Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.

Near-Infrared Activated Cyanine Dyes As Agents for Photothermal Therapy and Diagnosis of Tumors.

Today, it has become apparent that innovative treatment methods, including those involving simultaneous diagnosis and therapy, are particularly in demand in modern cancer medicine. The development of nanomedicine offers new ways of increasing the therapeutic index and minimizing side effects. The development of photoactivatable dyes that are effectively absorbed in the first transparency window of biological tissues (700–900 nm) and are capable of fluorescence and heat generation has led to the emergence of phototheranostics, an approach that combines the bioimaging of deep tumors and metastases and their photothermal treatment. The creation of near-infrared (NIR) light-activated agents for sensitive fluorescence bioimaging and phototherapy is a priority in phototheranostics, because the excitation of drugs and/or diagnostic substances in the near-infrared region exhibits advantages such as deep penetration into tissues and a weak baseline level of autofluorescence. In this review, we focus on NIR-excited dyes and discuss prospects for their application in photothermal therapy and the diagnosis of cancer. Particular attention is focused on the consideration of new multifunctional nanoplatforms for phototheranostics which allow one to achieve a synergistic effect in combinatorial photothermal, photodynamic, and/or chemotherapy, with simultaneous fluorescence, acoustic, and/or magnetic resonance imaging.

Integrated structural modeling and super-resolution imaging resolve GPCR oligomers.

Formation of G protein-coupled receptors (GPCRs) dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signaling activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, in both health and disease, from the study of this unique and complex subfamily of GPCRs with light on the luteinizing hormone receptor (LHR). Focus will be put on the results of an approach relying on the combination of atomistic modeling by protein-protein docking with super-resolution imaging. The latter could resolve single LHR molecules to ~8nm resolution in functional asymmetric dimers and oligomers, using dual-color photoactivatable dyes and localization microscopy (PD-PALM). Structural modeling of functionally asymmetric LHR trimers and tetramers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

Switcheroo yields photoactivatable dyes

A simple new method generates fluorescent dyes of many colors that can be activated by visible light. The approach provides a new set of tools for visualizing how proteins and other molecules behave in living cells without damaging tissue (J. Am. Chem. Soc. 2019, DOI: 10.1021/jacs.9b06237). Most existing photoactivatable dyes are bulky, so they can’t label smaller molecules with good resolution. And they need to be activated with high-energy laser light, which damages DNA and mitochondria, causes proteins to cross-link, and can bleach the dyes’ fluorescence. Broadening the palette of dyes that can be triggered with low-power visible light would be a boon for biological imaging in living tissue. In the new study, Han Xiao of Rice University and his team used a thionating agent called Lawesson’s reagent to switch an oxygen atom in an ordinary, nonphotoactivatable fluorescent dye to sulfur, essentially suppressing the glow. Unleashing the fluorescence simply involves

Single-Atom Fluorescence Switch: A General Approach toward Visible-Light-Activated Dyes for Biological Imaging.

Photoactivatable fluorophores afford powerful molecular tools to improve the spatial and temporal resolution of subcellular structures and dynamics. By performing a single sulfur-for-oxygen atom replacement within common fluorophores, we have developed a facile and general strategy to obtain photoactivatable fluorogenic dyes across a broad spectral range. Thiocarbonyl substitution within fluorophores results in significant loss of fluorescence via a photoinduced electron transfer-quenching mechanism as suggested by theoretical calculations. Significantly, upon exposure to air and visible light residing in their absorption regime (365-630 nm), thio-caged fluorophores can be efficiently desulfurized to their oxo derivatives, thus restoring strong emission of the fluorophores. The effective photoactivation makes thio-caged fluorophores promising candidates for super-resolution imaging, which was realized by photoactivated localization microscopy (PALM) with low-power activation light under physiological conditions in the absence of cytotoxic additives (e.g., thiols, oxygen scavengers), a feature superior to traditional PALM probes. The versatility of this thio-caging strategy was further demonstrated by multicolor super-resolution imaging of lipid droplets and proteins of interest.

Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy.

Super-resolution imaging has provided unprecedented insight in the molecular complexities of fundamental cell biological questions. For G protein-coupled receptors (GPCRs), its application to the study of receptor homomers and heteromers have unveiled the diversity of complexes these GPCRs can form at the plasma membrane at a structural and functional level. Here, we describe our methodological approach of photoactivated localization microscopy with photoactivatable dyes (PD-PALM) to visualize and quantify the spatial assembly of GPCR heteromers at the plasma membrane.

A volumetric three-dimensional digital light photoactivatable dye display

Volumetric three-dimensional displays offer spatially accurate representations of images with a 360° view, but have been difficult to implement due to complex fabrication requirements. Herein, a chemically enabled volumetric 3D digital light photoactivatable dye display (3D Light PAD) is reported. The operating principle relies on photoactivatable dyes that become reversibly fluorescent upon illumination with ultraviolet light. Proper tuning of kinetics and emission wavelengths enables the generation of a spatial pattern of fluorescent emission at the intersection of two structured light beams. A first-generation 3D Light PAD was fabricated using the photoactivatable dye N-phenyl spirolactam rhodamine B, a commercial picoprojector, an ultraviolet projector and a custom quartz imaging chamber. The system displays a minimum voxel size of 0.68 mm3, 200 μm resolution and good stability over repeated ‘on-off’ cycles. A range of high-resolution 3D images and animations can be projected, setting the foundation for widely accessible volumetric 3D displays.

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50μM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR.

Imaging Nanostructures by Single-Molecule Localization Microscopy in Organic Solvents.

The introduction of super-resolution fluorescence microscopy (SRM) opened an unprecedented vista into nanoscopic length scales, unveiling a new degree of complexity in biological systems in aqueous environments. Regrettably, supramolecular chemistry and material science benefited far less from these recent developments. Here we expand the scope of SRM to photoactivated localization microscopy (PALM) imaging of synthetic nanostructures that are highly dynamic in organic solvents. Furthermore, we characterize the photophysical properties of commonly used photoactivatable dyes in a wide range of solvents, which is made possible by the addition of a tiny amount of an alcohol. As proof-of-principle, we use PALM to image silica beads with radii close to Abbe's diffraction limit. Individual nanoparticles are readily identified and reliably sized in multicolor mixtures of large and small beads. We further use SRM to visualize nm-thin yet μm-long dynamic, supramolecular polymers, which are among the most challenging molecular systems to image.

Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling.