Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples.     

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.     

Intracellular Thiols and Photo‐Illumination Sequentially Activate Doubly Locked Molecular Probes for Long‐Term Cell Highlighting and Tracking with Precise Spatial Accuracy

A novel photoconvertible fluorescent probe, which can be activated by intracellular thiols, has been synthesized. Such a molecular probe comprises three parts: a 7‐aminocoumarin phototrigger, a thiol‐removable energy acceptor, and a caged fluorescein scaffold with intracellular thiols reactivity as the fluorescent reporter. Extracellularly, the energy acceptor blocks the emission of the coumarin that regulates the photocleavage and photoactivation of the fluorescein. Intracelluarly, the high concentration of thiols releases the energy acceptor, thus activating the S1 state of the phototrigger, which emits coumarin blue fluorescence for pre‐visualization and liberates the caged green‐fluorescent fluorescein to highlight the specific cell upon illumination. Compared to traditional photoactivated organic dyes, the intracellular thiols activated probe requires double activations: one by intracellular thiols and the other by light activation. The dual activations restrict fluorescence precisely inside live cells and at the particular spatial region of light activation, thus a probe with precise spatial accuracy in live cells.     

A Small‐Molecule Probe for Selective Profiling and Imaging of Monoamine Oxidase B Activities in Models of Parkinson’s Disease

The design of the first dual‐purpose activity‐based probe of monoamine oxidase B (MAO‐B) is reported. This probe is highly selective towards MAO‐B, even at high MAO‐A expression levels, and could sensitively report endogenous MAO‐B activities by both in situ proteome profiling and live‐cell bioimaging. With a built‐in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO‐B imaging probes failed to do thus far: the live‐cell imaging of MAO‐B activities without encountering diffusion problems.     

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.     

A Fluorescent Activatable AND‐Gate Chemokine CCL2 Enables In Vivo Detection of Metastasis‐Associated Macrophages

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND‐gates since they emit only after receptor binding and intracellular activation, showing enhanced selectivity over existing agents. We have applied this strategy to produce mCCL2‐MAF as the first probe for in vivo detection of metastasis‐associated macrophages in a preclinical model of lung metastasis. This strategy will accelerate the preparation of new chemokine‐based probes for imaging immune cell function in tumours.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

Application of porous metal enrichment probe sampling to single cell analysis using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF‐MS)

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix‐assisted laser desorption ionization time of flight mass spectrometry ( MALDI‐TOF‐MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self‐made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI‐TOF‐MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd.     

A Small‐Molecule FRET Reporter for the Real‐Time Visualization of Cell‐Surface Proteolytic Enzyme Functions

Real‐time imaging of cell‐surface‐associated proteolytic enzymes is critical to better understand their performances in both physiological and pathological processes. However, most current approaches are limited by their complexity and poor membrane‐anchoring properties. Herein, we have designed and synthesized a unique small‐molecule fluorescent probe, which combines the principles of passive exogenous membrane insertion and Förster resonance energy transfer (FRET) to image cell‐surface‐localized furin‐like convertase activities. The membrane‐associated furin‐like enzymatic cleavage of the peptide probe leads to an increased fluorescence intensity which was mainly localized on the plasma membrane of the furin‐expressed cells. This small‐molecule fluorescent probe may serve as a unique and reliable reporter for real‐time visualization of endogenous cell‐surfaceassociated proteolytic furin‐like enzyme functions in live cells and tissues using one‐photon and two‐photon microscopy.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.     

Environment‐sensitive Fluorescent Turn‐on Chemical Probe for the Specific Detection of O‐Methylguanine‐DNA Methyltransferase (MGMT) in Living Cells

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled daily at the hospitals, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this paper, we describe a MGMT‐activated fluorescence turn‐on probe for the rapid no‐wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O⁶‐benzyl‐guanine and an environment‐sensitive fluorophore, SBD. In the presence of MGMT, the enzyme transfers SBD to the protein active site where the hydrophobic surrounding causes the fluorophore to exhibit more than 50‐fold fluorescence enhancement. With this probe, bright fluorescence was observed for MGMT‐positive, Hela S3 and MCF‐7 cells, while MGMT‐deficient CHO cells displayed no fluorescence. We believe that this fluorescence activation probe design can also be extended to detect other transferases, for which there are still no effective methods to image them in living cells.     

A Reversible Fluorescent Probe for Real‐Time Live‐Cell Imaging and Quantification of Endogenous Hydropolysulfides

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their “intact” function in living cells has been limited owing to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe that reversibly and selectively reacts to hydropolysulfides. The probe enables live‐cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real‐time reversible monitoring of oxidative‐stress‐induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution on the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides.     

Polynuclear SmIII Polyamidoamine‐Based Dendrimer: A Single Probe for Combined Visible and Near‐Infrared Live‐Cell Imaging

We report herein the synthesis of a luminescent polynuclear dendritic structure (Sm^III‐G3P‐2,3Nap) in which eight Sm^III ions are sensitized by thirty‐two 2,3‐naphthalimide chromophores. Upon a single excitation wavelength, the dendrimer complex exhibits two types of emission in the visible and in the near‐infrared (NIR) ranges. Sm^III‐G3P‐2,3Nap was non‐cytotoxic after 24 h of incubation and up to 2.5 μM . The ability of the Sm^III‐based probe to be taken up by cells was confirmed by confocal microscopy. Epifluorescence microscopy validated Sm^III‐G3P‐2,3Nap as a versatile probe, capable of performing interchangeably in the visible or NIR for live‐cell imaging. As both emissions are obtained from a single complex, the cytotoxicity and biodistribution are inherently the same. The possibility for discriminating the sharp Sm^III signals from autofluorescence in two spectral ranges increases the reliability of analysis and reduces the probability of artifacts and instrumental errors.     

Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal‐Complex‐Based DNA Imaging Probes

The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir^IIIRu^II tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru^IIRu^II analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell‐free studies reveal that the Ir^IIIRu^II complex binds bovine serum albumin, BSA, in Sudlow’s Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum‐free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Synthesis and Systematic Evaluation of Dark Resonance Energy Transfer (DRET)‐Based Library and Its Application in Cell Imaging

In this paper, we report a new strategy for constructing a dye library with large Stokes shifts. By coupling a dark donor with BODIPY acceptors of tunable high quantum yield, a novel dark resonance energy transfer (DRET)‐based library, named BNM , has been synthesized. Upon excitation of the dark donor ( BDN ) at 490 nm, the absorbed energy is transferred to the acceptor ( BDM ) with high efficiency, which was tunable in a broad range from 557 nm to 716 nm, with a high quantum yield of up to 0.8. It is noteworthy to mention that the majority of the non‐radiative energy loss of the donor was converted into the acceptor’s fluorescence output with a minimum leak of donor emission. Fluorescence imaging tested in live cells showed that the BNM compounds are cell‐permeable and can also be employed for live‐cell imaging. This is a new library which can be excited through a dark donor allowing for strong fluorescence emission in a wide range of wavelengths. Thus, the BNM library is well suited for high‐throughput screening or multiplex experiments in biological applications by using a single laser excitation source.     

Dual‐Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time‐Resolved Imaging Independent of Probe Pretreatment and Probe Concentration

Developing luminescent probes with long lifetime and high emission efficiency is essential for time‐resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation‐induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid‐and‐flexible alternation design in donor–acceptor–donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual‐phase strong and long‐lived emission allows a time‐resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.