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The measurement of ion concentrations and fluxes inside living cells is key to understanding cellular physiology. Fluorescent indicators that can infiltrate and provide intel on the cellular environment are critical tools for biological research. Developing these molecular informants began with the seminal work of Racker and colleagues ( Biochemistry (1979) 18, 2210), who demonstrated the passive loading of fluorescein in living cells to measure changes in intracellular pH. This work continues, employing a mix of old and new tradecraft to create innovative agents for monitoring ions inside living systems.

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo. A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

Visualizing neuronal activity in vivo Imaging the changes in fluorescence of voltage-sensitive reagents would enable monitoring of the activity of neurons in vivo. Abdelfattah et al. created such a voltage indicator by designing a protein that combines the voltage sensor domain from microbial rhodopsin with a domain that captures a dye molecule with exceptional brightness and photostability. When the protein was expressed in mice, flies, or zebrafish, they could monitor single action potentials in dozens of neurons simultaneously for many minutes. Science , this issue p. 699

Development of single-molecule localization microscopy (SMLM) has sparked a revolution in biological imaging, allowing "super-resolution" fluorescence microscopy below the diffraction limit of light. The past decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-molecule fluorescent probes for this technique. In this review, written by chemists for chemists, we detail the history of single-molecule localization microscopy and collate the collection of probes with demonstrated utility in SMLM. We hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biological samples with exquisite detail.

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure–activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. Vibrio cholerae esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from Mycobacterium tuberculosis displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr-119 in ybfF and His-187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multifaceted future applications, including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

SUMMARY Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure., In Brief Yan et al. examine how functional activity of nicotinic cholinergic receptors is distributed in diverse VTA cell types, revealing nAChR activity in VTA glutamate neurons. These receptors modulate local glutamate transmission in VTA, suggesting mechanisms by which nicotine influences mesolimbic circuitry.

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca

Photolabile protecting groups (or “photocages”) enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input–output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca2+‐sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca2+]. Caging a fluorophore with this ion‐sensitive moiety yields an “ion integrator” that permanently marks cells undergoing high Ca2+ flux during an illumination‐defined time period. Our general design concept demonstrates a new class of light‐sensitive material for cellular imaging, sensing, and targeted molecular delivery.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1. Fil: Swinstead, Erin E.. National Institutes of Health; Estados Unidos Fil: Miranda, Tina B.. National Institutes of Health; Estados Unidos Fil: Paakinaho, Ville. National Institutes of Health; Estados Unidos Fil: Baek, Songjoon. National Institutes of Health; Estados Unidos Fil: Goldstein, Ido. National Institutes of Health; Estados Unidos Fil: Hawkins, Mary. National Institutes of Health; Estados Unidos Fil: Karpova, Tatiana S.. National Institutes of Health; Estados Unidos Fil: Ball, David. National Institutes of Health; Estados Unidos Fil: Mazza, Davide. Universita Vita-salute San Raffaele; Italia Fil: Lavis, Luke D.. Howard Hughes Medical Institute; Estados Unidos Fil: Grimm, Jonathan B.. Howard Hughes Medical Institute; Estados Unidos Fil: Morisaki, Tatsuya. National Institutes of Health; Estados Unidos. State University of Colorado - Fort Collins; Estados Unidos Fil: Grøntved, Lars. National Institutes of Health; Estados Unidos Fil: Presman, Diego Martin. National Institutes of Health; Estados Unidos Fil: Hager, Gordon L.. National Institutes of Health; Estados Unidos

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.

Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen.

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the “photon budget” require better and brighter fluorophores, causing a renewed interest in synthetic dyes. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical fluorophores to modern biological imaging.

Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology—the melding of old and new chemical knowledge—leading to useful molecules for advanced biochemical and biological experiments.

An efficient, one-step, and highly functional group-compatible synthesis of substituted N-aryl-2H-indazoles is reported via the rhodium(III)-catalyzed C–H bond addition of azobenzenes to aldehydes. The regioselective coupling of unsymmetrical azobenzenes was further demonstrated and led to the development of a new removable aryl group that allows for the preparation of indazoles without N-substitution. The 2-aryl-2H-indazole products also represent a new class of readily prepared fluorophores for which initial spectroscopic characterization has been performed.

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.

Bovine pancreatic ribonuclease (RNase A) can enter human cells, even though it lacks a cognate cell-surface receptor protein. Here, we report on the biochemical basis for its cellular uptake. Analyses in vitro and in cellulo revealed that RNase A interacts tightly with abundant cell-surface proteoglycans containing glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, as well as with sialic acid-containing glycoproteins. The uptake of RNase A correlates with cell anionicity, as quantified by measuring electrophoretic mobility. The cellular binding and uptake of RNase A contrast with those of Onconase®, an amphibian homologue that does not interact tightly with anionic cell-surface glycans. As anionic glycans are especially abundant on human tumor cells, our data predicate utility for mammalian ribonucleases as cancer chemotherapeutic agents.

p-Nitrophenyl acetate is the most commonly used substrate for detecting thecatalytic activity of esterases, including those that activate prodrugs in human cells. Thissubstrate is unstable in aqueous solution, limiting its utility. Here, a stable chromogenicsubstrate for esterases is produced by the structural isolation of an acetyl ester andp-nitroaniline group using a trimethyl lock moiety. Upon ester hydrolysis, unfavorablesteric interactions between the three methyl groups of this o-hydroxycinnamic acidderivative encourage rapid lactonization to form a hydrocoumarin and releasep-nitroaniline. This “prochromophore†could find use in a variety of assays.

Fluorescent molecules are indispensable tools in modern biochemical and biological research, being used as labels for biomolecules, indicators for ions, stains for organelles, and substrates for enzymes.[1] The major targets of this last class are hydrolases that catalyze the removal of a masking moiety, thereby modulating fluorescence.[2] A critical property of fluorogenic hydrolase substrates is their chemical stability in aqueous solution, as spontaneous hydrolysis can compete deleteriously with enzymatic activity. New substrate classes that exhibit increased stability while maintaining enzymatic reactivity would be highly desirable. Our laboratory recently reported on the use of the “trimethyl lock” strategy in the design of a latent fluorophore.[3] This latent fluorophore consists of a trimethyl lock component inserted between a dye and enzyme-reactive group. The trimethyl lock is an o-hydroxycinnamic acid derivative in which unfavorable steric interactions between three methyl groups encourage rapid lactonization to form a hydrocoumarin and release a leaving group.[4, 5] Our initial latent fluorophore exhibited remarkable stability in aqueous solution, but released a xanthene dye (rhodamine 110) upon hydrolytic cleavage by an esterase. Here, we explore the modularity of our design, probing its applicability to unrelated dyes that absorb at short (blue) and long (red) wavelengths. Coumarin-based compounds comprise an important class of blue dyes possessing UV or near-UV excitation wavelengths.[6] Acyl esters and acyloxymethyl ethers of 7-hydroxycoumarin (i.e., umbelliferone) can be substrates for esterases.[7–9] 7-Amino-4-methylcoumarin (AMC) is used widely as the basis for protease substrates.[10–12] Upon amidation, the excitation and emission wavelengths of AMC are shifted to shorter wavelengths with concomitant reduction in quantum yield.[10] We reasoned that AMC could be subjected to our latent fluorophore strategy. Accordingly, we condensed AMC with acetylated trimethyl lock 1[13] to give pro-fluorophore 2 (Scheme 1), which displayed the expected hypsochromic shift of excitation and emission spectra relative to free AMC (Figure 1). The hydrolysis of profluorophore 2 was catalyzed by porcine liver esterase (PLE) with kcat/KM = 2.5 × 105 M−1s−1 and KM = 8.2 μM (Figure 2A). This kcat/KM value is 102-fold greater than the apparent kcat/KM value for the latent fluorophore based on rhodamine 110.[3] (We use the term “apparent” because full fluorescence manifestation requires the lactonization of two trimethyl lock moieties.[3]) Figure 1 Normalized fluorescent excitation–emission spectra of pro-fluorophore 2 and AMC. Figure 2 (a) Kinetic traces (λex 365 nm, λem 460 nm) and Michaelis–Menten plot (inset) for the hydrolysis of pro-fluorophore 2 (20 μM→9.8 nM) by PLE (2.5 μg/mL); kcat/KM = 2.5 × 105 M−1s−1 ... Scheme 1 Route for the synthesis of pro-fluorophore 2, and the chemical structure of 4-methylumbelliferyl acetate (3). To evaluate the relative stability of pro-fluorophore 2, we monitored the accretion of fluorescence in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA; 1.0 mg/mL) and either pro-fluorophore 2 or 4-methylumbelliferyl acetate (3, which is a common esterase substrate[9]). Pro-fluorophore 2 proved to be highly stable compared to 4-methylumbelliferyl acetate (Figure 3). An additional advantage of pro-fluorophore 2 is that the product of its hydrolysis, AMC, shows little change in fluorescence at pH ≥ 5.[1] In contrast, the fluorescence of 4-methylumbelliferone is highly variable due to its pKa = 7.40[14] being near the physiological pH. Figure 3 Time course of the generation of fluorescence (λex 365 nm, λem 460 nm) from pro-fluorophore 2 (50 nM) or methylumbelliferyl acetate 3 (50 nM) in PBS containing BSA (1 mg/mL). To access longer wavelengths, we turned to the oxazine class of red dyes. One such dye, cresyl violet (CV) has been used for decades to stain tissues[15, 16] and as a laser dye.[17] Although CV has a maximal absorbance at 586 nm, its fused benzo ring broadens its absorption spectrum[18] and thereby allows excitation with a variety of light sources. Diamide derivatives of CV are virtually nonfluorescent and thus useful in the production of fluorogenic protease substrates.[19–21] The attributes of CV make this dye an attractive candidate for our latent fluorophore strategy. Accordingly, we condensed CV with acetylated trimethyl lock 1[13] to give pro-fluorophore 4 (Scheme 2). Although pro-fluorophore 4 was stable in PBS containing BSA (data not shown), its hydrolysis was catalyzed by PLE with apparent kinetic parameters of kcat/KM = 1.2 × 105 M−1s−1 and KM = 0.14μM (Figure 2B). The kcat/KM value for the hydrolysis of pro-fluorophore 4 is similar to that of pro-fluorophore 2; the KM value is, however, 10-fold lower than that of pro-fluorophore 2. Scheme 2 Route for the synthesis of pro-fluorophore 4. Pro-fluorophore 4 serves as a probe for esterase activity in live mammalian cells. The broad excitation peak allowed confocal microscopy experiments using excitation at 543 nm and emission at 605 nm. Pro-fluorophore 4 was hydrolyzed by esterases endogenous in the cells of a rodent (Figure 4A) or human (Figure 4B) to give a red-stained cytosol within minutes. The somewhat enhanced fluorescence observed in the human cells could be indicative of more efficient internalization or hydrolysis of the latent fluorophore. After longer incubation, the liberated CV became further localized in subcellular compartments (data not shown), which is consistent with previous reports.[21] To date, there have been few reports of long-wavelength esterase substrates that are useful in cell biology.[1] Its evident stability, optical properties, and fast intracellular eduction make pro-fluorophore 4 useful in a wide variety of biological applications, especially in assays involving fluorophores of different wavelengths. Figure 4 Unwashed mammalian cells incubated for 15 min with pro-fluorophore 4 (10 μM) at 37 °C in DMEM and counter-stained with Hoechst 33342 (5% v/v CO2(g), 100% humidity). (a) CHO K1 cells. (b) HeLa cells. In conclusion, we have established that one component of our latent fluorophores—the dye—is modular (Figure 5). Specifically, we have now prepared useful latent fluorophores from three dyes (blue, green,[3] and red), all linked by a trimethyl lock moiety to an esterase-reactive group. In future work, we shall explore the modularity of the other component—the enzyme-reactive group. We anticipate that the end result will be a broad spectrum of stable latent fluorophores with numerous applications in biochemical and biological research. Figure 5 Modules in the latent fluorophores described in this work.

The evolutionary rate of proteins involved in obligate protein–protein interactions is slower and the degree of co-evolution higher than that for non-obligate protein–protein interactions. The co-evolution of the proteins involved in certain non-obligate interactions is, however, essential to cell survival. To gain insight into the co-evolution of one such non-obligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.