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Marine organisms are a rich source of bioactive secondary metabolites. Although many marine natural products with bioactivities have been isolated, successful elucidation of their mechanisms of action remains limited. In this study, we prepared a probe molecule based on the marine cyclic peptide kapakahine A (1) by introducing a linker with an azide terminal group, which enables the introduction of fluorescent groups for the effective monitoring of subcellular localization, or coupling to affinity beads for the pull-down of target proteins. The results of LC/MS/MS measurements, ProteinPilot analysis, and Western blotting suggest that kapakahine A interacts with the mitochondrial inner membrane proteins PHB1, PHB2, and ANT2, which is consistent with the results of the subcellular localization analysis using a fluorescent probe.

A single-wavelength genetically encoded sensor of extracellular glutamate is reported. The sensor—iGluSnFR—is bright and photostable under both one- and two-photon illumination and is shown to work for in vivo imaging in worms, zebrafish and mice. We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ , and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone. Control of molecular architecture using this technology has allowed us to address the long-standing technical issue of contact quenching between fluorescent dyes offering new possibilities in the life sciences arena. Much like peptidic sequences coding for enzymes, cofactors, and receptors (all needing control of tertiary structure for proper function via primary sequence) our programmable system approaches a similar endpoint using a phosphate based polymeric backbone assembled in a completely automated fashion. Using this novel technology, we have efficiently synthesized several types of fluorescent dyes and demonstrated the programmability in molecule design, including the increases in brightness of the fluorescence emission.

In this study, we report the synthesis and the photochemical behavior of a series of new \"click-on\" fluorescent probes designed to detect singlet oxygen. They include a highly fluorescent chemical structure, an aryloxazole ring, linked to a furan moiety operating as singlet oxygen trap. Their activity depends on both the structure of the aryloxazole fluorophore and the electron-donating and electron-accepting properties of the substituents attached to the C-5 of the furan ring. All probes are selectively oxidized by singlet oxygen to give a single fluorescent product in methanol and produce negligible amounts of singlet oxygen themselves by self-sensitization. The most promising dyad, (E)-2-(2-(5-methylfuran-2-yl)vinyl)naphtho[1,2-d]oxazole, FN-6, shows outstanding reactivity and sensitivity: it traps singlet oxygen with a rate constant (5,8 ± 0.1) x 1(07) M-1 s-1 and its fluorescence increases by a factor of 500 upon reaction. Analysis of the dyads reactivity in terms of linear free energy relationships using the modified Swain and Lupton parameter F and the Fukui condensed function for the electrophilic attack, suggests that cycloaddition of singlet oxygen to the furan ring is partially concerted and possibly involves an exciplex with a \"more open\" structure than could be expected for a concerted cycloaddition.

Key Points Fluorescence imaging can transform the way surgeries are performed, through the intraoperative identification of vital structures, lymph nodes and cancer in real time Near-infrared (NIR) fluorescence is particularly advantageous for use in clinical settings owing to improved depth penetration and low autofluorescence in the NIR wavelength range compared with shorter wavelengths Many targeted NIR fluorophores are currently in preclinical development; however, no cancer-targeted NIR fluorophores or devices for intraoperative NIR fluorescence detection of cancer have received commercial approval for human use Multiple early phase clinical trials are underway to evaluate targeted fluorophores for real-time, intraoperative cancer detection in humans The use of targeted fluorophores for the intraoperative detection of cancer might improve survival rates and functional outcomes in patients with cancer Currently, substantial regulatory challenges and clinical trial considerations constitute barriers for the adoption of fluorescence-guided surgery in clinical settings Intraoperative fluorescence enables highly specific real-time detection of tumours at the time of surgery. In particular, near-infrared (NIR) fluorescence is a promising tool currently being tested in clinical settings. Zhang et al . discuss the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, as well as the challenges associated with their effective application in clinical settings. Over the past two decades, synergistic innovations in imaging technology have resulted in a revolution in which a range of biomedical applications are now benefiting from fluorescence imaging. Specifically, advances in fluorophore chemistry and imaging hardware, and the identification of targetable biomarkers have now positioned intraoperative fluorescence as a highly specific real-time detection modality for surgeons in oncology. In particular, the deeper tissue penetration and limited autofluorescence of near-infrared (NIR) fluorescence imaging improves the translational potential of this modality over visible-light fluorescence imaging. Rapid developments in fluorophores with improved characteristics, detection instrumentation, and targeting strategies led to the clinical testing in the early 2010s of the first targeted NIR fluorophores for intraoperative cancer detection. The foundations for the advances that underline this technology continue to be nurtured by the multidisciplinary collaboration of chemists, biologists, engineers, and clinicians. In this Review, we highlight the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, and consider the unique challenges associated with their effective application in clinical settings.

Background It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. Results We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). Conclusions Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the ‘lowest’ and the ‘highest’ levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo) . By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

In this work, a novel ratiometric fluorescent sensor, based on carbon dots (CDs) and gold nanoclusters (AuNCs), is developed for highly sensitive and selective visual colorimetric detection of Cu2+ and alkaline phosphatase (ALP). The ratiometric fluorescent sensor was synthesized by covalently linking 11-mercaptoundecanoic acid (11-MUA)–stabilized AuNCs to the surface of amino-functionalized CD/SiO2 nanoparticles. The red fluorescence of the AuNCs can be quenched by Cu2+ owing to coordination between Cu2+ and 11-MUA; however, the blue emission of the CDs was insensitive to Cu2+ owing to the protective silica shell. The quenching of the AuNCs’ fluorescence returned when PPi was added because of the higher affinity between Cu2+ and PPi than that between Cu2+ and 11-MUA. In the presence of ALP, PPi was catalytically hydrolyzed into phosphate (Pi), which showed a much weaker affinity for Cu2+. Thus, Cu2+ ions were released, and the fluorescence of the AuNCs was quenched once more. Based on this principle, Cu2+ and ALP could be simultaneously detected. The developed ratiometric fluorescent sensor could detect Cu2+ over a range from 0.025 to 4 μM with a detection limit of 0.013 μM and ALP over a range from 0.12 to 15 U/L with a detection limit of 0.05 U/L. The present method was successfully applied for the detection of Cu2+ and ALP in real water samples and in human serum samples, respectively. This ratiometric fluorescent approach may provide a highly sensitive and accurate platform for visual Cu2+ and ALP sensing in environmental monitoring and medical diagnosis.

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns. Fruit flies and other animals have complex body plans containing many different types of cells. To make and maintain these body plans, individual genes must be switched on and off at specific times in particular cells to control how the animal grows. Some of these genes may be switched on for long periods of time, while others may be rapidly switched on and off on repeated occasions. Fluorescent reporter proteins have been extensively used to study gene activity in cells. Typically, this involves linking the gene encoding the fluorescent reporter to a gene of interest, so that when the gene is switched on a fluorescent protein will also be produced. The fluorescent protein emits light of a particular color and measuring this light provides a way to monitor a gene’s activity. Unfortunately, fluorescent proteins tend to break down slowly, and the level of fluorescence emitted cannot fluctuate quickly enough to reflect rapid changes in gene behavior. One way to overcome this limitation is to use destabilized fluorescent proteins that degrade more rapidly inside cells. However, current strategies for creating these proteins cause them to emit less light, making fluorescence more difficult to detect. To address this issue, He et al. developed a new green destabilized protein, adding elements that increase production of the protein so a greater amount of light can be emitted. The green destabilized protein was then combined with a red fluorescent reporter that degrades more slowly to develop a new tool called TransTimer. When the gene linked to the reporter switches on, the green destabilized protein turns on before the red reporter turns on. But, as the gene switches off, the destabilized protein will degrade until only the red signal remains. This allows the ratio of green to red color emitted from the TransTimer to indicate the timing of a gene’s activity. Using this tool, He et al. uncovered new details about the patterns of activity of two signals, known as Notch and STAT, that were largely missed by studies using traditional fluorescent reporters. Further experiments demonstrated that TransTimer can be used to carry out large-scale screens in living fruit flies, which have not been possible with more time consuming live-cell imaging techniques. The fluorescent reporter developed by He et al. will be a useful tool to understand when and where genes are switched on during the lives of fruit flies. In the future, TransTimer could be adapted for use in other model animals or plants.

Despite the growing popularity of blood oxygen level-dependent (BOLD) functional MRI (fMRI), understanding of its underlying principles is still limited. This protocol describes a technique for simultaneous measurement of neural activity using fluorescent calcium indicators together with the corresponding hemodynamic BOLD fMRI response in the mouse brain. Our early work using small-molecule fluorophores in rats gave encouraging results but was limited to acute measurements using synthetic dyes. Our latest procedure combines fMRI with optical detection of cell-type-specific virally delivered GCaMP6, a genetically encoded calcium indicator (GECI). GCaMP6 fluorescence, which increases upon calcium binding, is collected by a chronically implanted optical fiber, allowing longitudinal studies in mice. The chronic implant, placed horizontally on the skull, has an angulated tip that reflects light into the brain and is connected via fiber optics to a remote optical setup. The technique allows access to the neocortex and does not require adaptations of commercial MRI hardware. The hybrid approach permits fiber-optic calcium recordings with simultaneous artifact-free BOLD fMRI with full brain coverage and 1-s temporal resolution using standard gradient-echo echo-planar imaging (GE-EPI) sequences. The method provides robust, cell-type-specific readouts to link neural activity to BOLD signals, as emonstrated for task-free ('resting-state') conditions and in response to hind-paw stimulation. These results highlight the power of fiber photometry combined with fMRI, which we aim to further advance in this protocol. The approach can be easily adapted to study other molecular processes using suitable fluorescent indicators.