A Baldwin-favored Cyclization Inspires the Development of Fluorogenic Polymethine Dyes for Bioimaging.

Fluorescence imaging is an invaluable tool to study biological processes, and fluorogenic dyes are crucial to enhance cell permeability and target intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. Here, we present a general strategy to transform polymethine compounds into fluorogenic dyes by implementing a 5-exo-trig ring-closure. This method provides access to bright, fluorogenic polymethine dyes with emissions across the visible and near-infrared spectrum.

A Far-Red Fluorescent Probe to Visualize Gram-Positive Bacteria in Patient Samples.

Gram-positive bacteria, in particular Staphylococcus aureus (S. aureus), are the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and high mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red fluorescent probe to detect Gram-positive bacteria, with a focus on staphylococci, in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging.

Fluorogenic polymethine dyes by intramolecular cyclization.

Fluorescence imaging plays a pivotal role in the study of biological processes, and cell-permeable fluorogenic dyes are crucial to visualize intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. In this review, we summarize recent advances in the development of fluorogenic polymethine dyes via intramolecular cyclization. Finally, we offer an outlook on the prospects of fluorogenic polymethine dyes for bioimaging.

A general strategy to develop fluorogenic polymethine dyes for bioimaging.

Fluorescence imaging is an invaluable tool to study biological processes and further progress depends on the development of advanced fluorogenic probes that reach intracellular targets and label them with high specificity. Excellent fluorogenic rhodamine dyes have been reported, but they often require long and low-yielding syntheses, and are spectrally limited to the visible range. Here we present a general strategy to transform polymethine compounds into fluorogenic dyes using an intramolecular ring-closure approach. We illustrate the generality of this method by creating both spontaneously blinking and no-wash, turn-on polymethine dyes with emissions across the visible and near-infrared spectrum. These probes are compatible with self-labelling proteins and small-molecule targeting ligands, and can be combined with rhodamine-based dyes for multicolour and fluorescence lifetime multiplexing imaging. This strategy provides access to bright, fluorogenic dyes that emit at wavelengths that are more red-shifted compared with those of existing rhodamine-based dyes.

A locally activatable sensor for robust quantification of organellar glutathione.

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signalling pathways. Achieving a detailed understanding of intracellular GSH homeostasis depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Here we present a GSH-sensing platform for live-cell imaging, termed targetable ratiometric quantitative GSH (TRaQ-G). This chemogenetic sensor possesses a unique reactivity turn-on mechanism, ensuring that the small molecule is only sensitive to GSH in a desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein to give a ratiometric response. Using TRaQ-G fused to a redox-insensitive fluorescent protein, we demonstrate that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. This sensor was used in combination with a redox-sensitive fluorescent protein to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.

A live-cell image-based machine learning strategy for reducing variability in PSC differentiation systems.

The differentiation of pluripotent stem cells (PSCs) into diverse functional cell types provides a promising solution to support drug discovery, disease modeling, and regenerative medicine. However, functional cell differentiation is currently limited by the substantial line-to-line and batch-to-batch variabilities, which severely impede the progress of scientific research and the manufacturing of cell products. For instance, PSC-to-cardiomyocyte (CM) differentiation is vulnerable to inappropriate doses of CHIR99021 (CHIR) that are applied in the initial stage of mesoderm differentiation. Here, by harnessing live-cell bright-field imaging and machine learning (ML), we realize real-time cell recognition in the entire differentiation process, e.g., CMs, cardiac progenitor cells (CPCs), PSC clones, and even misdifferentiated cells. This enables non-invasive prediction of differentiation efficiency, purification of ML-recognized CMs and CPCs for reducing cell contamination, early assessment of the CHIR dose for correcting the misdifferentiation trajectory, and evaluation of initial PSC colonies for controlling the start point of differentiation, all of which provide a more invulnerable differentiation method with resistance to variability. Moreover, with the established ML models as a readout for the chemical screen, we identify a CDK8 inhibitor that can further improve the cell resistance to the overdose of CHIR. Together, this study indicates that artificial intelligence is able to guide and iteratively optimize PSC differentiation to achieve consistently high efficiency across cell lines and batches, providing a better understanding and rational modulation of the differentiation process for functional cell manufacturing in biomedical applications.

Single-Molecule Peptide Identification Using Fluorescence Blinking Fingerprints.

The ability to identify peptides with single-molecule sensitivity would lead to next-generation proteomics methods for basic research and clinical applications. Existing single-molecule peptide sequencing methods can read some amino acid sequences, but they are limited in their ability to distinguish between similar amino acids or post-translational modifications. Here, we demonstrate that the fluorescence intermittency of a peptide labeled with a spontaneously blinking fluorophore contains information about the structure of the peptide. Using a deep learning algorithm, this single-molecule blinking pattern can be used to identify the peptide. This method can distinguish between peptides with different sequences, peptides with the same sequence but different phosphorylation patterns, and even peptides that differ only by the presence of epimerized residues. This study builds the foundation for a targeted proteomics method with single-molecule sensitivity.

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells.

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

Long-Term, Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores.

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing.

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

Disruption of mitochondrial redox homeostasis by enzymatic activation of a trialkylphosphine probe.

Redox homeostasis is essential for cell function and its disruption is associated with multiple pathologies. Redox balance is largely regulated by the relative concentrations of reduced and oxidized glutathione. In eukaryotic cells, this ratio is different in each cell compartment, and disruption of the mitochondrial redox balance has been specifically linked to metabolic diseases. Here, we report a probe that is selectively activated by endogenous nitroreductases, and releases tributylphosphine to trigger redox stress in mitochondria. Mechanistic studies revealed that, counterintuitively, release of a reducing agent in mitochondria rapidly induced oxidative stress through accumulation of superoxide. This response is mediated by glutathione, suggesting a link between reductive and oxidative stress. Furthermore, mitochondrial redox stress activates a cellular response orchestrated by transcription factor ATF4, which upregulates genes involved in glutathione catabolism.

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.

Dual-Activatable Cell Tracker for Controlled and Prolonged Single-Cell Labeling.

Cell trackers are fluorescent chemical tools that facilitate imaging and tracking cells within live organisms. Despite their versatility, these dyes lack specificity, tend to leak outside of the cell, and stain neighboring cells. Here, we report a dual-activatable cell tracker for increased spatial and temporal staining control, especially for single-cell tracking. This probe overcomes the typical problems of current cell trackers: off-target staining, high background signal, and leakage from the intracellular medium. Staining with this dye is not cytotoxic, and it can be used in sensitive primary cells. Moreover, this dye is resistant to harsh fixation and permeabilization conditions and allows for multiwavelength studies with confocal microscopy and fluorescence-activated cell sorting. Using this cell tracker, we performed in vivo homing experiments in mice with primary splenocytes and tracked a single cell in a heterogeneous, multicellular culture environment for over 20 h. These experiments, in addition to comparative proliferation studies with other cell trackers, demonstrated that the signal from this dye is retained in cells for over 72 h after photoactivation. We envision that this type of probes will facilitate the analysis of single-cell behavior and migration in cell culture and in vivo experiments.

Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery.

Chemical biologists have developed many tools based on genetically encoded macromolecules and small, synthetic compounds. The two different approaches are extremely useful, but they have inherent limitations. In this Minireview, we highlight examples of strategies that combine both concepts to tackle challenging problems in chemical biology. We discuss applications in imaging, with a focus on super-resolved techniques, and in probe and drug delivery. We propose future directions in this field, hoping to inspire chemical biologists to develop new combinations of synthetic and genetically encoded probes.

The Bioorthogonal Isonitrile-Chlorooxime Ligation.

Bioorthogonal reactions are valuable tools for the selective labeling and imaging of natural products and proteins. Here, we present the reaction between isonitriles and chlorooximes as a ligation that proceeds quickly (k ≈ 1 M-1 s-1) and with high chemoselectivity in an aqueous environment. Imaging of metabolically labeled cell surface glycans underlined the tolerance of the ligation to common functional groups in cellular systems. Live-cell dual-labeling experiments revealed that the isonitrile-chlorooxime ligation is orthogonal to the strain-promoted azide-alkyne cycloaddition.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases Using a Photo-Crosslinking Fluorescent Sensor.

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines, and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, three-dimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.

Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching.

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.