A Strategy for Specific Fluorescence Imaging of Monoamine Oxidase A in Living Cells

Monoamine oxidase (MAO) has two isoforms, MAO‐A and MAO‐B, which show different functions, and thus selective fluorescence imaging is important for biological studies. Currently, however, specific detection of MAO‐A remains a great challenge. Herein, we report a new strategy for specific imaging of MAO‐A through the design of fluorogenic probes combining the characteristic structure of an inhibitor of the target enzyme along with propylamine as a recognition moiety. The high specificity of our representative probe is demonstrated by imaging MAO‐A in different live cells such as SH‐SY5Y (high levels of MAO‐A) and HepG2 (high levels of MAO‐B), and further validated by western blot analyses. The superior specificity of the probe may enable the accurate detection of MAO‐A in complex biosystems. Importantly, the use of the characteristic structure of an inhibitor, as demonstrated in this work, may serve as a general strategy to design specific recognition moieties for fluorogenic probes for enzymes.     

Rational Design of a Two‐Photon Fluorogenic Probe for Visualizing Monoamine Oxidase A Activity in Human Glioma Tissues

Monoamine oxidases have two functionally distinct but structurally similar isoforms (MAO‐A and MAO‐B). The ability to differentiate them by using fluorescence detection/imaging technology is of significant biological relevance, but highly challenging with available chemical tools. Herein, we report the first MAO‐A‐specific two‐photon fluorogenic probe ( F1 ), capable of selective imaging of endogenous MAO‐A enzymatic activities from a variety of biological samples, including MAO‐A‐expressing neuronal SY‐SY5Y cells, the brain of tumor‐bearing mice and human Glioma tissues by using two‐photon fluorescence microscopy (TPFM) with minimal cytotoxicity.     

Photoelectrocyclization as an Activation Mechanism for Organelle‐Specific Live‐Cell Imaging Probes

Photoactivatable fluorophores are useful tools in live‐cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle‐specific live‐cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water‐soluble, non‐cytotoxic, cell‐permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre‐activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single‐cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single‐ or multi‐cell labeling experiments.     

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.     

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.     

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.     

Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

Until recently, chemiluminescence cell images could only be obtained using luciferase‐activated probes. Moreover, chemiluminescence microscopy cell‐imaging has not been demonstrated for natively expressed enzymes like cathepsin B. Herein, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for the detection and imaging of cathepsin B. The probe activation mechanism relies on the release of a dioxetane intermediate, which undergoes chemiexcitation to emit green light with high efficiency under physiological conditions. Using the probe, we obtained clear images of cancerous leukemia and colon cells. This is the first demonstration of chemiluminescence cell images obtained by a probe for a natively expressed endogenous enzyme. We anticipate that the concept presented in this study will be broadly used to develop analogous probes for other important proteases relevant to biomolecular processes.     

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs)

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.     

Tetraphenylethylene Conjugated with a Specific Peptide as a Fluorescence Turn‐On Bioprobe for the Highly Specific Detection and Tracing of Tumor Markers in Live Cancer Cells

Smart molecular probes and flexible methods are attracting remarkable interest for the visualization of cancer‐related biological and chemical events. In this work, a new fluorescence turn‐on probe with dual‐recognition characteristics for the specific imaging of cancer cells is reported. This new bioprobe is rationally designed by linking tetraphenylethylene (TPE), an aggregation‐induced emission (AIE) fluorophore, with the small peptide IHGHHIISVG (referred to as AP2H), a targeting ligand to the broad‐spectrum cancer‐related protein LAPTM4B. The binding of the probe TPE‐AP2H with the target, both in solution and at the cellular level, switches on the fluorescence of TPE because of the inhibition of internal rotations within the TPE framework. Accordingly, this bioprobe allows the real‐time monitoring and subcellular localization of LAPTM4B in cancer cells, with a very high target‐to‐background ratio for the imaging. Furthermore, brighter fluorescence images are detected after incubation of TPE‐AP2H with tumor cells at lower pH values. Thus, this new bioprobe is more advantageous because it can simultaneously target the LAPTM4B protein and sense the characteristic low‐pH environment of tumor cells. In addition, TPE‐AP2H displays high photostability and low cytotoxicity. Therefore, this new bioprobe is promising for the more accurate and reliable imaging of tumor markers in live cancer cells.     

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non‐engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene‐specific RNA aptamer, combined with a cell‐permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live‐cell imaging of the endogenous mRNA of β‐actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin‐2, cortactin, and cytoplasmic FMR1‐interacting protein 2. The RNA‐imaging technology and its further optimization might permit live‐cell imaging of any RNA molecules.     

Live‐Cell Stimulated Raman Scattering Imaging of Alkyne‐Tagged Biomolecules

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.     

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.     

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.     

Highly Selective Mitochondrial Targeting by a Ruthenium(II) Peptide Conjugate: Imaging and Photoinduced Damage of Mitochondrial DNA

Mitochondrial DNA (mtDNA) plays a crucial but incompletely understood role in cellular biochemistry and etiology of numerous disease states. Thus, there is an urgent need for targeted probes that can dynamically respond to changes to mtDNA such as copy number in live cells, but it is difficult to permeate the mitochondrial membrane of the living cell. Now, a ruthenium(II) light‐switching probe targeted by peptide vectorization selectively to mitochondrial nucleoids is presented. Evidence for DNA binding by the probe in live cells is derived from confocal fluorescence microscopy, resonance Raman, and luminescence lifetime imaging. While viable under imaging conditions, specific staining of mitochondrial DNA permitted efficient and selective photoinduced toxicity on a cell‐by‐cell basis under higher excitation intensities. This powerful combination of imaging and photocytotoxicity is an important step towards realizing phototheranostic application of such Ru^II probes.     

An AIE‐Based Probe for Rapid and Ultrasensitive Imaging of Plasma Membranes in Biosystems

The abnormality of the plasma membrane (PM) is an important biomarker for cell status and many diseases. Hence, visualizing the PM, especially in complex systems, is an emerging field in the life sciences, especially in low‐resource settings. Herein, we developed a water‐soluble PM‐specific probe utilizing electrostatic and hydrophobic interaction strategies with aggregation‐induced emission as the signal output. The probe could image the PM with many advanced features (wash‐free, ultrafast staining process, excellent PM specificity, and good biocompatibility), which were demonstrated by the PM imaging of neurons. The probe allowed for the first time the imaging of erythrocytes in the complex brain environment through a fluorescence‐based method. Moreover, the PM of the epidermal and partial view of the eyeball structure of live zebrafish are also revealed.     

In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live‐Cell Imaging Probes

In spite of the wide application potential of 1,2,4,5‐tetrazines, particularly in live‐cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)‐3‐substituted 6‐alkenyl‐1,2,4,5‐tetrazine derivatives through an elimination–Heck cascade reaction. By using this strategy, we provide 24 examples of π‐conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta‐1,3‐diene‐substituted tetrazines, and a diverse array of fluorescent probes suitable for live‐cell imaging. These highly conjugated probes show very strong fluorescence turn‐on (up to 400‐fold) when reacted with dienophiles such as cyclopropenes and trans‐cyclooctenes, and we demonstrate their application for live‐cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live‐cell imaging.     

A Ratiometric Two‐Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In Cellulo Observation of Lysosomal Membrane Fusion Processes

Vesicles exchange their contents through membrane fusion processes, kiss‐and‐run and full‐collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two‐photon probe offering real‐time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two‐photon live‐cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss‐and‐run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full‐fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small‐molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.     

Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H₂S level in cells. The two sites of the probe reactive to H₂S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H₂S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H₂S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H₂S in cellular signaling and in various human diseases.     

NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling

To address existing limitations in live neuron imaging, we have developed NeuO , a novel cell‐permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.