Photoactivatable Fluorophores Research Articles

Modulating the photolysis of aryl azides in a supramolecular host to develop photoactivatable fluorophores.

Photolysis of aryl azides is a convenient method to approach more functionalized systems in chemical biology. Here, we present a set of photoactivatable aryl azides that undergo controlled reaction pathways within the cucurbit[7]uril (CB7) cavity upon photolysis. The fluorescence turn-on process is utilized for bioimaging.

A Blue Light and Two-Photon Activatable Rhodamine Fluorophore.

Photoactivatable fluorophores (PAFs) are powerful tools for biological imaging applications because they provide spatiotemporal control of fluorescence distribution. Many of the existing PAFs can only be activated by UV irradiation. In our study, we present a blue light (1P) and NIR light (2P) activatable rhodamine fluorophore. Next to the description of the synthesis and the investigation of the photoreaction, we demonstrate the use of our PAF in the context of laser scanning microscopy. By immobilization of our PAF in a hydrogel, we were able to write and read spatially resolved illumination patterns with excellent contrast after both one-photon and two-photon excitation.

Photoactivatable BODIPYs for Live-Cell PALM.

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

Photoactivatable Fluorophores for Bioimaging Applications.

Photoactivatable fluorophores provide the opportunity to switch fluorescence on exclusively in a selected area within a sample of interest at a precise interval of time. Such a level of spatiotemporal fluorescence control enables the implementation of imaging schemes to monitor dynamic events in real time and visualize structural features with nanometer resolution. These transformative imaging methods are contributing fundamental insights on diverse cellular processes with profound implications in biology and medicine. Current photoactivatable fluorophores, however, become emissive only after the activation event, preventing the acquisition of fluorescence images and, hence, the visualization of the sample prior to activation. We developed a family of photoactivatable fluorophores capable of interconverting between emissive states with spectrally resolved fluorescence, instead of switching from a nonemissive state to an emissive one. We demonstrated that our compounds allow the real-time monitoring of molecules diffusing across the cellular blastoderm of developing embryos as well as of polymer beads translocating along the intestinal tract of live nematodes. Additionally, they also permit the tracking of single molecules in the lysosomal compartments of live cells and the visualization of these organelles with nanometer resolution. Indeed, our photoactivatable fluorophores may evolve into invaluable analytical tools for the investigation of the fundamental factors regulating the functions and structures of cells at the molecular level.

Direct optical resolution in the intra-molecular distance range.

Estimations of molecular distances <10 nm have long been the almost exclusive domain of Förster resonance energy transfer (FRET) and related spectroscopic techniques. By site-specifically labeling molecular subunits with (at least) two fluorophores, single-molecule FRET has also allowed for the monitoring of distances within macromolecules. In this presentation, we report on the direct determination of such intra-molecular distances based on the MINFLUX localization concept. We measured intra-macromolecular spacings of photoactivatable small-molecule fluorophores in well-characterized uni- or few-molecular systems, with simple polypeptides and proteins serving as initial examples. Our experiments indicate that the widely employed STORM-type dark-state photoblinking of cyanines reaches an impasse for dye separations below ∼6-10 nm, as the dyes couple and no longer behave independently. The complex photophysics of such dye-dye communication impedes the reliable, isolated detection of their signals. As we show however, sequential photoactivation - providing the required off-on-off transitions in a more orthogonal way - is a viable strategy to read out dye coordinates even for separations as low as few nm, beyond any perceived remaining barriers of resolution. Our results, with direct resolution of intra-molecular distances and conformations, herald the possibility of fluorescence-based analyses of structural biology at room or physiological temperature relying on direct positional measurements rather than on indirect FRET readouts.

A Universal Photoactivatable Tag Attached to Fluorophores Enables Their Use for Single-Molecule Imaging.

Single molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3-dihydro-1,4-oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODIPY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time-lapse super-resolution tracking in living cells and super-resolution imaging of microtubule structures in neurons.

A Universal Photoactivatable Tag Attached to Fluorophores Enables Their Use for Single‐Molecule Imaging

AbstractSingle molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3‐dihydro‐1,4‐oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODIPY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time‐lapse super‐resolution tracking in living cells and super‐resolution imaging of microtubule structures in neurons.

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.

Rational design of state-depending photoactivatable and photoconvertible fluorescent AIEgens through a rapid photocyclodehydrogenation reaction

Photosensitive fluorophores have grand value and bright prospective in diverse photo-controlled applications including super-resolution imaging and advanced patterning; however, photoactivatable and photoconvertible small fluorophores encounter great challenges in comparison to their fast-growing protein-based counterparts. Herein, we proposed a general design strategy to achieve state-depending photoactivatable and photoconvertible fluorophores based on an AIE-active molecular skeleton through introducing a dipyridinylethene unit which easily undergoes a rapid photocyclodehydrogenation reaction. It is found that introducing a photoactive dipyrinylethene unit into an AIE-active tetraarylethene molecule can effectively regulate its dominant photophysical emission and photochemical reaction in a controllable way, and these combined compounds show predominant aggregation-induced emission properties in solid state but exhibit dominant photoactivatable and photoconvertible fluorescence behaviours in dispersed state. The photoactivatable and photoconvertible fluorescence is attributed to the rapid photo-induced cyclodehyrogenation reaction and AIE nature of the species involved in the photochemical processes. These findings outline a general design strategy to achieve photoactivatable and photoconvertible fluorescence systems by introducing a specific subunit easily undergoing rapid and efficient photocyclodehyrogenation reaction, and demonstrate exciting use of these state-dependent photosensitive fluorophores in diverse photo-controlled practical applications.

Photoactivatable Fluorescent Tags for Dual-Modality Positron Emission Tomography Optical Imaging.

Fluorescent protein conjugates are vital tools in a wide range of scientific disciplines from basic biochemical research to applications in clinical pathology and intraoperative surgery. We report the synthesis and characterization of photoactivatable fluorophores (PhotoTags) based on the functionalization of coumarin, fluorescein, BODIPY, rhodamine B, and cyanine dyes with a photochemically active aryl azide group. Photochemical labeling experiments using human serum albumin produced fluorescent proteins in high yields under irradiation with ultraviolet light for <15 min. We also synthesized DFO-RhodB-PEG3-ArN3─a photoactivatable compound that can be radiolabeled with 89Zr for applications in optical imaging and positron emission tomography. One-pot 89Zr-radiolabeling and light-induced protein conjugation produced [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab. Proof-of-concept studies in vitro and in vivo confirmed that [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab is a potential dual-modality agent for detecting human epidermal growth factor receptor 2 (HER2/neu) expression. Overall, the PhotoTag technology represents a rapid, synthetically versatile, and user-friendly approach for generating novel protein conjugates.

A tetrazole-ene photoactivatable fluorophore with improved brightness and stability in protic solution

The pyrazoline fluorophore, generated by photoinduced tetrazole-ene cycloaddition, shows faint fluorescence in protic solvents. To suppress this fluorescence-quenching, we rationally designed a series of substituted diaryl tetrazoles at the N-side phenyl ring to produce a tetrazole-ene based photoactivatable fluorophore. Spectroscopic and cellular imaging studies demonstrated that the version of the fluorophore with a bis(trifluoromethyl)benzene substituent exhibited significantly enhanced brightness and photostability.

BODIPYs with Photoactivatable Fluorescence.

The borondipyrromethene (BODIPY) chromophore is a versatile platform for the construction of photoresponsive dyes with unique properties. Specifically, its covalent connection to a photocleavable group can be exploited to engineer compounds with photoswitchable fluorescence. The resulting photoactivatable fluorophores can increase their emission intensity or shift their emission wavelengths in response to switching. Such changes permit the spatiotemporal control of fluorescence with optical stimulations and the implementation of imaging strategies that would be impossible to replicate with conventional fluorophores. Indeed, BODIPYs with photoactivatable fluorescence enable the selective highlighting of intracellular targets, the nanoscaled visualization of sub-cellular components, the real-time monitoring of dynamic events and the photochemical writing of optical barcodes.

Oxime as a general photocage for the design of visible light photo-activatable fluorophores.

Photoactivatable fluorophores have been widely used for tracking molecular and cellular dynamics with subdiffraction resolution. In this work, we have prepared a series of photoactivatable probes using the oxime moiety as a new class of photolabile caging group in which the photoactivation process is mediated by a highly efficient photodeoximation reaction. Incorporation of the oxime caging group into fluorophores results in loss of fluorescence. Upon light irradiation in the presence of air, the oxime-caged fluorophores are oxidized to their carbonyl derivatives, restoring strong fluorophore fluorescence. To demonstrate the utility of these oxime-caged fluorophores, we have created probes that target different organelles for live-cell confocal imaging. We also carried out photoactivated localization microscopy (PALM) imaging under physiological conditions using low-power light activation in the absence of cytotoxic additives. Our studies show that oximes represent a new class of visible-light photocages that can be widely used for cellular imaging, sensing, and photo-controlled molecular release.

Site-Specific Incorporation of a Photoactivatable Fluorescent Amino Acid.

Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

PhotoTag: Photoactivatable Fluorophores for Protein Labeling.

We report experimental studies on the development of photoactivatable fluorophores for rapid, light-induced synthesis of protein conjugates. Proof-of-concept studies demonstrated that electronic excitation of photoactivatable BODIPY-ArN3 (1) in the presence of different proteins leads to efficient labeling in less than 10 min. After synthesis and isolation of the fluorescently tagged protein, photochemical conversion yields using human serum albumin and onartuzumab were 47 ± 7% and 42 ± 5%, respectively.

Live-Cell Imaging at the Nanoscale with Bioconjugatable and Photoactivatable Fluorophores.

Optical diffraction fundamentally limits the spatial resolution of conventional fluorescence images to length scales that are, at least, 2 orders of magnitude longer than the dimensions of individual molecules. As a result, the development of innovative probes and imaging schemes to overcome diffraction is very much needed to enable the investigation of the fundamental factors regulating cellular functions at the molecular level. In this context, the chemical synthesis of molecular constructs with photoactivatable fluorescence and the ability to label subcellular components of live cells can have transformative implications. Indeed, the fluorescence of the resulting assemblies can be activated with spatiotemporal control, even in the intracellular environment, to permit the sequential localization of individual emissive labels with precision at the nanometer level and the gradual reconstruction of images with subdiffraction resolution. The implementation of these operating principles for subdiffraction imaging, however, is only possible if demanding photochemical and photophysical requirements to enable photoactivation and localization as well as stringent structural requisites to allow the covalent labeling of intracellular targets in live cells are satisfied. Because of these complications, only a few synthetic photoactivatable fluorophores with appropriate performance for live-cell imaging at the nanoscale have been developed so far. Significant synthetic efforts in conjunction with spectroscopic analyses are still very much needed to advance this promising research area further and turn photoactivatable fluorophores into the imaging probes of choice for the investigation of live cells.

A Synthetic Strategy for the Structural Modification of Photoactivatable BODIPY‐Oxazine Dyads

AbstractStrategies to regulate the ability of photoactivatable fluorophores to absorb activating photons are essential to allow their operation under illumination conditions compatible with biological samples. In this context, our laboratories identified a viable synthetic protocol to introduce a conjugated substituent on the photocleavable component of a BODIPY‐oxazine dyad and enhance its molar absorption coefficient at the activation wavelength. This structural transformation translates into a four‐fold absorbance increase in the spectral window appropriate for photoactivation and does not prevent the photochemical transformation. Indeed, the photoinduced cleavage of the oxazine component occurs in organic solvents, aqueous solutions and hydrogel matrices with a concomitant bathochromic shift in BODIPY emission. These results demonstrate that the photophysical properties of this family of BODIPY‐oxazine dyads can be manipulated with the aid of chemical synthesis without suppressing their photochemical behavior.

Tetrazine as a general phototrigger to turn on fluorophores.

Light-activated fluorescence affords a powerful tool for monitoring subcellular structures and dynamics with enhanced temporal and spatial control of the fluorescence signal. Here, we demonstrate a general and straightforward strategy for using a tetrazine phototrigger to design photoactivatable fluorophores that emit across the visible spectrum. Tetrazine is known to efficiently quench the fluorescence of various fluorophores via a mechanism referred to as through-bond energy transfer. Upon light irradiation, restricted tetrazine moieties undergo a photolysis reaction that generates two nitriles and molecular nitrogen, thus restoring the fluorescence of fluorophores. Significantly, we find that this strategy can be successfully translated and generalized to a wide range of fluorophore scaffolds. Based on these results, we have used this mechanism to design photoactivatable fluorophores targeting cellular organelles and proteins. Compared to widely used phototriggers (e.g., o-nitrobenzyl and nitrophenethyl groups), this study affords a new photoactivation mechanism, in which the quencher is photodecomposed to restore the fluorescence upon light irradiation. Because of the exclusive use of tetrazine as a photoquencher in the design of fluorogenic probes, we anticipate that our current study will significantly facilitate the development of novel photoactivatable fluorophores.

Photoactivation of silicon rhodamines via a light-induced protonation

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.