Mayeul Collot

Living cells of brown algae are difficult to observe in 3D because pigments such as fucoxanthin and chlorophyll diffract light. Furthermore, at the beginning of their life, brown algae develop slowly in seawater. To gain insight into the 3D shape and size of brown algal cells during embryogenesis, we designed a fluorescence probe that labels the plasma membrane efficiently and selectively. Styryl benzoindoleninium sulfonate (SBIS) is a bright orange fluorogenic probe that is soluble and virtually non-emissive in seawater and is activated upon binding to the plasma membrane. Unlike Calcofluor White, SBIS enables observation of cells at thicknesses of up to 25 µm. More importantly, SBIS allows 3D observation of the cells in the growing uniseriate filaments of Ectocarpus sp., the polystichous filaments of Sphacelaria rigidula and the cellular monolayered lamina of Saccharina latissima over periods of up to 7 days. Altogether, these properties allow visualization of entire cell contours in living brown algae, making the study of early development at the cellular level in 4D now possible in these marine organisms.

Super‐resolution imaging based on the localization of single emitters requires a spatio‐temporal control of the ON and OFF states. To this end, photoactivatable fluorophores are adapted as they can be turned on upon light irradiation. Here, we present a concept called self‐triggered photooxidation cascade (STPC) based on the photooxidation of a plasma membrane‐targeted leuco‐rhodamine (LRhod‐PM), a non‐fluorescent reduced form of a rhodamine probe. Upon visible light irradiation the small number of oxidized rhodamines, Rhod‐PM, acts as a photosensitizer to generate singlet oxygen capable of oxidizing the OFF state LRhod‐PM thereby switching it to its ON state. We showed that this phenomenon is kinetically favored by a high local concentration and propagates quickly when the probe is embedded in membrane bilayers. In addition, we showed that the close proximity of the dyes favors the photobleaching. At the single‐molecule level, the concomitant activation/bleaching phenomena allow reaching a single‐molecule blinking regime enabling single‐molecule localization microscopy for super‐resolution of live cellular membranes and their thin processes including filopodia and tuneling nanotubes.

Characterization of nanoscale formulations is a continuous challenge. Size, morphology and surface properties are the most common characterizations. However, physicochemical properties inside the nanoparticles, like viscosity, cannot be directly measured. Herein, we propose an original approach to measuring dynamic viscosity using a lipidic molecular rotor solubilized in the core of nano-formulations. These molecules undergo conformational changes in response to viscosity variations, leading to observable changes in fluorescence intensity and lifetime, able to sense the volume properties of dispersed nanodomains. The lipophilic molecular rotor (BOPIDY derivatives) was specifically synthesized and characterized as oil viscosity sensing in large volumes. A second part of the study compares these results with rBDP-Toco in nano-emulsions. The objective is to evaluate the impact of the formulation, droplet size and composition on the viscosity of the droplet's core. The lipophilic rotor showed a universal behavior whatever the oil composition, giving a master curve. Applied to nano-formulations, it reveals the viscosity inside the nanoemulsion droplets, enabling the detection of slight variations between reference oil samples and the nano-formulated ones. This new tool opens the way to the fine characterization of complex colloids and multi-domain nano and micro systems, potentially applied to hybrid materials and biomaterials.

We have developed spontaneously blinking fluorescent probes based on the reversible spirolactamization of rhodamine, to efficiently image the plasma membrane (PM) of live cells with enhanced resolution using SMLM. This study demonstrates that the blinking efficiency of spiroamide PM probes is not solely governed by their pKa; the presence of a charged polar group on the amide should also be taken into account.

The rise of colistin-resistant <i>Acinetobacter baumannii</i> has severely limited treatment options for infections caused by this pathogen. While terpene alcohols and fatty acids have shown potential to enhance colistin’s efficacy, but their high lipophilicity limits their clinical application. To address this, we developed water-dispersible lipid nanoparticles (LNPs) in two sizes (40 nm and 130 nm), loaded with these compounds to act as colistin adjuvants. Among eleven LNP formulations, six significantly reduced colistin’s minimum inhibitory concentration (MIC) by 16- to 64-fold. The most effective, featuring (E,E)-farnesol and myristic acid, were further examined for bactericidal activity, membrane disruption, cytotoxicity, and <i>in vivo</i> efficacy in <i>Galleria mellonella</i> larvae. Time-kill studies demonstrated that at an adjuvant concentration of 60 mg/L, these LNPs eradicated bacteria when combined with 4 mg/L free colistin for resistant isolates (MIC = 128 mg/L) and 0.06 mg/L for susceptible isolates (MIC = 0.5 mg/L), without regrowth. Myristic acid-loaded LNPs combined with free colistin at 1/8 MIC resulted in a 4.2-fold higher mortality rate than the combination with (E,E)-farnesol-loaded LNPs in resistant strains. This result was correlated with a 45-fold faster increase in inner membrane permeability, measured by propidium iodide (PI) uptake, in the presence of myristic acid-loaded LNPs compared with a 13-fold faster increase with (E,E)-farnesol-loaded LNPs. DiSC3(5) assays revealed that LNPs alone depolarised the bacterial inner membrane, with enhanced effects when combined with colistin at 1/8 MIC, a result not observed with colistin alone at this concentration. As with PI uptake, this inner membrane depolarising effect was more pronounced with myristic acid-loaded LNPs than with (E,E)-farnesol-loaded LNPs in resistant strains, suggesting that the colistin adjuvant effect of these lipophilic compounds is due to their ability to help colistin destabilise the bacterial inner membrane. Cytotoxicity assays demonstrated no adverse effects on bone marrow macrophages after 6 h of exposure, although some toxicity was observed after 24 h. No mortality was observed in <i>Galleria mellonella</i> larvae over 7 days following three consecutive days of treatment with colistin and LNPs. Notably, the combination of (E,E)-farnesol-loaded LNPs and colistin significantly improved the survival of <i>Galleria</i> infected with <i>A. baumannii</i>. These results suggest that lipophilic-adjuvant-loaded LNPs may offer a promising strategy to enhance colistin efficacy and combat antibiotic-resistant <i>A. baumannii</i> infections.

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green‐emitting photoactivatable furanyl‐BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo‐oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93‐fold fluorescence enhancement at 510 nm, 49% yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super‐resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells.

Abstract Photomodulable fluorophores constitute advanced materials as they possess the ability to modify their photophysical properties upon photoirradiation. A new mechanism of photoconversion is recently established, called Directed Photooxidation Induced Conversion based on the coupling of fluorophores with Aromatic Singlet oxygen Reactive Moieties (ASORMs). In this work, The Directed Photooxidation Induced Conversion (DPIC) mechanism is intended to be applied to Perylene BisImide (PBI) due to its appealing photophysical properties. The experimental results showed that coupling two ASORMs to the PBI core, here furan and pyrrole, led to impressive photomodulable fluorophores. While PBI‐F exhibited a photoconversion of 100 nm shift, PBI‐P displayed an 80‐fold fluorescence intensity enhancement upon photoactivation. Analysis of the photoproducts showed that the conversion do not involve an addition of singlet oxygen on the ASORM. Instead, photoconversion occurred through efficient successive photocyclizations. Finally, intracellular vesicles are successfully photoconverted by means of endocytosed PLGA‐polymer nanoparticles loaded with PBI‐F. This study highlights the unique capability of furan‐ and pyrrole‐conjugated fluorophores to enable advanced optical materials with phototransformation properties.

Neurovascular coupling (NVC), which mediates rapid increases in cerebral blood flow in response to neuronal activation, is commonly used to map brain activation or dysfunction. Here we tested the reemerging hypothesis that CO2 generated by neuronal metabolism contributes to NVC. We combined functional ultrasound and two-photon imaging in the mouse barrel cortex to specifically examine the onsets of local changes in vessel diameter, blood flow dynamics, vascular/perivascular/intracellular pH, and intracellular calcium signals along the vascular arbor in response to a short and strong CO2 challenge (10 s, 20%) and whisker stimulation. We report that the brief hypercapnia reversibly acidifies all cells of the arteriole wall and the periarteriolar space 3–4 s prior to the arteriole dilation. During this prolonged lag period, NVC triggered by whisker stimulation is not affected by the acidification of the entire neurovascular unit. As it also persists under condition of continuous inflow of CO2, we conclude that CO2 is not involved in NVC.

Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter‐ and intra‐molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin‐membrane distance of 2.5 nm. We demonstrate that QD‐FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub‐nanometer accuracy.

Abstract Neurovascular coupling (NVC), which mediates rapid increases in cerebral blood flow in response to neuronal activation, is commonly used to map brain activation or dysfunction. Here we tested the reemerging hypothesis that CO 2 generated by neuronal metabolism contributes to NVC. We combined functional ultrasound and two-photon imaging in the mouse barrel cortex to specifically examine the onsets of local changes in vessel diameter, blood flow dynamics, vascular/perivascular/intracellular pH, and intracellular calcium signals along the vascular arbor in response to a short and strong CO 2 challenge (10 s, 20%) and whisker stimulation. We report that the brief hypercapnia reversibly acidifies all cells of the arteriole wall and the periarteriolar space 3–4 s prior to the arteriole dilation. During this prolonged lag period, NVC triggered by whisker stimulation is not affected by the acidification of the entire neurovascular unit. As it also persists under condition of continuous inflow of CO 2 , we conclude that CO 2 is not involved in NVC.

Abstract Fluorophores bearing cationic pendants, such as the pyridinium group, tend to preferentially accumulate in mitochondria, whereas those with pentafluorophenyl groups display a distinct affinity for the endoplasmic reticulum. In this study, we designed fluorophores incorporating pyridinium and pentafluorophenyl pendants and examined their impact on sub‐cellular localization. Remarkably, the fluorophores exhibited a notable propensity for the mitochondrial membrane. Furthermore, these fluorophores revealed dual functionality by facilitating the detection of viscosity changes within the sub‐cellular environment and serving as heavy‐atom‐free photosensitizers. With easy chemical tunability, wash‐free imaging, and a favorable signal‐to‐noise ratio, these fluorophores are valuable tools for imaging mitochondria and investigating their cellular processes.

Although amphiphilic cyclodextrin derivatives (ACDs) serve as valuable building blocks for nanomedicine formulations, their widespread production still encounters various challenges, limiting large-scale manufacturing. This work focuses on a robust alternative pathway using mineral base catalysis to transesterify β-cyclodextrin with long-chain vinyl esters, yielding ACD with modular and controlled hydrocarbon chain grafting. ACDs with a wide range of degrees of substitution (DS) were reliably synthesized, as indicated by extensive physicochemical characterization, including MALDI-TOF mass spectrometry. The influence of various factors, including the type of catalyst and the length of the hydrocarbon moiety of the vinyl ester, was studied in detail. ACDs were assessed for their ability to form colloidal suspensions by nanoprecipitation, with or without PEGylated phospholipid. Small-angle X-ray scattering and cryo-electron microscopy revealed the formation of nanoparticles with distinct ultrastructures depending on the DS: an onion-like structure for low and very high DS, and reversed hexagonal organization for DS between 4.5 and 6.1. We confirmed the furtivity of the PEGylated versions of the nanoparticles through complement activation experiments and that they were well tolerated in-vivo on a zebrafish larvae model after intravenous injection. Furthermore, a biodistribution experiment showed that the nanoparticles left the bloodstream within 10 h after injection and were phagocytosed by macrophages.

Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.

Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

Three new amphiphilic molecules were synthetized on a basis of a common monomer, octadecyl succinic anhydride (OSA), on which were grafted different PEGylated hydrophilic polar head. The objective of this study is to investigate a potential relationship between the chemical structure of the surfactant, and the efficiency to generate nano-emulsions through the spontaneous nano-emulsification method. Beyond the innovative synthesis and comparison of these amphiphiles, a comprehensive comparison is suggested, comparing size distribution and polydispersity for the different composition parameters, as well making a bridge with critical micelle concentration and hydrophile lipophile balance (HLB). Using OSA monomeric entity as common hydrophobic moiety, the variations in the surfactant molecules were done on the hydrophilic moiety, through the addition of one or two Jeffamine chains in different configurations, so-called C18⊖-PEG, C18-PEG and C18-PEG2. The results disclosed that C18⊖-PEG allows producing smallest size distribution and lowest PDI values. Moreover, C18⊖-PEG presents the highest critical micellar concentration, linked to a higher hydrophilicity of the molecule. This impacts the balance between surfactant affinity for oil and aqueous phases, a point probably related to the spontaneous emulsification efficiency. A last part of the study regarded the optimization of the emulsification efficiency, through a systematic study in ternary composition map, to disclosed that the best conditions are included in the lower surfactant concentrations, for water and oil contents higher and lower than 50%, respectively. The main idea behind this study was to bring further insights into the unclear relationship between the chemical structure of nonionic surfactants, and the efficiency of the emulsification by spontaneous low-energy method.

Polymeric nanoparticles (NPs) continue to assert their high potential as efficient nanovectors for nanomedicine and imaging agents. Among them, biocompatible polyester block copolymers (BCP) possess appealing features as their physicochemical properties can be readily modified by the composition of their blocks. Although the fluorescent labeling of polymers is often used to track the formed NPs in bioimaging, it is rarely used to characterize their physicochemical properties. In this work, we assumed that the branching degree of the hydrophobic block copolymer might play an important role in the properties of the biocompatible NPs and thus could help in controlling their cargo encapsulation and release. To this end, clickable PEG-PCL block copolymers with various branching degrees were synthesized. Owing to their covalent fluorescent labeling using strain-promoted azidealkyne cycloaddition, several parameters like the critical aggregation concentration of the BCPs as well as the colloidal stability, the core polarity, and stealth of the NPs have been studied. Taking advantage of the fluorescence labeling of the NPs' core, their ability to encapsulate and release a fluorescent cargo (Rhodamine C 18) was assessed by Forster resonance energy transfer (FRET). The observed differences in the release profile were confirmed in cells where the fluorescent NPs and their cargo were tracked. Finally, the cargo release of endocytosed NPs was imaged and assessed by FRET ratiometric imaging. This study proved that the covalent fluorescent labeling of the BCPs is an efficient tool, offering various methods to characterize and assess the effects of polymers' modifications on the NPs' properties.

Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.

Endothelial senescence has been identified as an early event in the development of endothelial dysfunction, a hallmark of cardiovascular disease. This study developed theranostic nanocarriers (NC) decorated with VCAM-1 antibodies (NC-VCAM-1) in order to target cell surface VCAM-1, which is overexpressed in senescent endothelial cells (ECs) for diagnostic and therapeutic purposes. Incubation of Ang IIinduced premature senescent ECs or replicative senescent ECs with NC-VCAM-1 loaded with lipophilic fluorescent dyes showed higher fluorescence signals than healthy EC, which was dependent on the NC size and VCAM-1 antibodies concentration, and not observed following masking of VCAM-1. NC loaded with omega 3 polyunsaturated fatty acid (NC-EPA:DHA6:1) were more effective than native EPA:DHA 6:1 to prevent Ang II-induced VCAM-1 and p53 upregulation, and SA-β-galactosidase activity in coronary artery segments. These theranostic NC might be of interest to evaluate the extent and localization of endothelial senescence and to prevent pro-senescent endothelial responses.

This review describes the different chemical approaches and strategies to fluorescently label block copolymers. The review also focuses on the properties of fluorescent markers and the bioimaging applications allowed by the labeling.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β-and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α-and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/ activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Visualization of sub-cellular organelles allows the determination of various cellular processes and the underlying mechanisms. Herein, we report a fluorescent probe, bearing push-pull substituents emitting at 600 nm and its application in cellular imaging. The probe shows dual imaging of mitochondria and nucleoli and maps mitochondrial viscosity in live cells under various physiological variations and show minimum cytotoxicity. Nucleolar staining is confirmed by RNAase digestion.

Fluorogenic molecular rotors have demonstrated their efficiency to probe viscous environments in various cell compartments. Green emitting BODIPY rotors are non-charged and thus are widely used in bioimaging. However, non-charged rotors operating in the near infrared region and exhibiting high brightness are rare. We herein synthesized five different merocyanines dyes (MCs) from a dihydroxanthene scaffold and studied their spectroscopic properties. Notable differences were observed, which allowed us to establish structure-properties relationships. Among MCs, MC-TB, bearing a thiobarbituric electron acceptor group, displayed high sensitivity to viscosity, excellent photostability, high extinction coefficient (4100 000 M À1 cm À1) and bright emission with sharp peaks in the near infrared region (B700 nm), which are favorable features for bioimaging. In cells, MC-TB stained the most hydrophobic and viscous organelles, namely the lipid droplets. To increase the versatility of this new rotor, a mitochondria targeted version, MC-TB-Mito, was synthesized. The latter showed similar appealing photophysical properties and it was successfully used to report variation and heterogeneity of mitochondrial viscosity using multicolor fluorescence microscopy and fluorescence lifetime imaging (FLIM). Finally, we used MC-TB-Mito to reveal an important increase of mitochondrial viscosity during cell apoptosis.

Nanoemulsions (NEs) are water-dispersed oil droplets that constitute stealth biocompatible nanomaterials. NEs can reach an impressive degree of fluorescent brightness owing to their oily core that can encapsulate a large number of fluorophores on the condition the latter are sufficiently hydrophobic and oil-soluble. BODIPYs are among the brightest green emitting fluorophores and as neutral molecules possess high lipophilicity. Herein, we synthesized three different natural lipid-BODIPY conjugates by esterification of an acidic BODIPY by natural lipids, namely: α-tocopherol (vitamin E), cholesterol, and stearyl alcohol. The new BODIPY conjugates were characterized in solvents and oils before being encapsulated in NEs at various concentrations. The physical (size, stability over time, leakage) and photophysical properties (absorption and emission wavelength, brightness, photostability) are reported and showed that the nature of the lipid anchor and the nature of the oil used for emulsification greatly influence the properties of the bright NEs.

Fluorescent materials are continuously contributing to important advances in the field of bioimaging. Among these materials, dioxaborine-based fluorescent materials (DBFM) are arousing growing interest. Due to their rigid structures conferred by a cyclic boron complex, DBFM possess appealing photophysical properties including high extinction coefficients and quantum yields as well as emission in the near infrared, enhanced photostability and high two-photon absorption. We herein discuss the recent advances of DBFM that found use in bioimaging applications. This review covers the development of fluorescent molecular probes for biomolecules (DNA, proteins), small molecules (cysteine, H 2 O 2 , oxygen), ions and the environment (polarity, viscosity) as well as polymers and nanomaterials used in bioimaging. This review aims at providing a comprehensive and critical insight on DBFM by highlighting the assets of these promising materials in bioimaging but also by pointing out their limitations that would require further developments.

Lipid nanoemulsions (NEs), owing to their controllable size (20 to 500 nm), stability and bio-compatibility, are now frequently used in various fields, such as food, cosmetics, pharmaceuticals, drug delivery, and even as nano-reactor for chemical synthesis. Moreover, being composed of components generally recognized as safe (GRAS), they can be considered as "green" nanoparticles that mimic closely lipoproteins and intracellular lipid droplets. Therefore, they attracted attention as carriers of drugs and fluorescent dyes for both bioimaging and studying the fate of nanoemulsions in cells and small animals. In this review, we describe the composition of dye-loaded NEs, methods for their preparation and emerging biological applications. We focus on design of bright fluorescent NEs with high dye loading and minimal aggregation-caused quenching (ACQ). Common issues including dye leakage and NEs stability are discussed, highlighting advanced techniques for their characterization, such as Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Attempts to functionalize NEs surface are also discussed. Then, biological applications for bioimaging and single-particle tracking in cell and small animals as well as biomedical applications for photodynamic therapy are described. Finally, challenges and future perspectives of fluorescent NEs are discussed.

Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.

Fluorogenic probes enable imaging biomolecular targets with high sensitivity and maximal signal-to-background ratio in nonwash conditions. Here, we focus on the molecular design of biotinylated dimeric squaraines that undergo aggregationcaused quenching in aqueous media through intramolecular H-type dimerization, but turn on their fluorescence in apolar environment due to target-mediated disaggregation. Our structure-properties study revealed that depending on the linkers used to connect the squaraine dyes, different aggregation patterns could be obtained (intramolecular dimerization versus intermolecular aggregation) leading to different probing efficiencies. Using a relatively short L-lysine linker we developed a bright fluorogenic probe, Sq2B, displaying only intramolecular dimerization-caused quenching properties in aqueous media. The latter was successfully applied for imaging biotin receptors, in particular sodium-dependent multivitamin transporter (SMVT), which are overexpressed at the surface of cancer cells. Competitive displacement with SMVT-targets, such as biotin, lipoic acid or sodium pantothenate, showed Sq2B targeting ability to SMVT. This fluorogenic probe for biotin receptors could distinguish cancer cells (HeLa and KB) from model non-cancer cell lines (NIH/3T3 and HEK293T). The obtained results provide guidelines for development of new dimerization-based fluorogenic probes and propose new bright tools for imaging biotin receptors, which is particularly important for specific detection of cancer cells.

Among the lipid nanoparticles, Lipid Polymer Hybrid Nanoparticles (HNPs) composed of an oily core and a polymeric shell, display interesting features as efficient drug carriers due to the high loading capability of the oil phase and the stability and surface functionalization of the polymer shell. Herein, we formulated lipid-core/polymer-shell hybrid nanoparticles (HNPs) by a simple nanoprecipitation method involving Vitamin E Acetate (VEA) as the oily core and a tailor-made amphiphilic polymer as a wrapping shell. The fluorescence labeling of the oil, using a newly developed green fluorogenic BODIPY tracker, and of the polymer using a covalent attachment of a red emitting rhodamine allowed to assessing the formation, the composition and the stability of these new hybrid nanoparticles using dual color electrophoresis gel analysis. This technique, combined to conventional DLS and electronic microscopy analysis, allowed us to quickly determine that 20 wt % of polymer was an optimal ratio for obtaining stable HNPs by nanoprecipiation. Finally, we showed that using different polymeric shells, various HNPs can be obtained and finely discriminated by combined electrophoresis and two-color labeling approach.

The plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell lines including neurons (primary culture and tissue imaging). Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Visualizing single organic nanoparticles (NPs) in vivo remains a challenge, which could greatly improve our understanding of the bottlenecks in the field of nanomedicine. To achieve high single-particle fluorescence brightness, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with a bulky hydrophobic counterion (perfluorinated tetraphenylborate) as a fluorophore insulator to prevent aggregation-caused quenching. To create NPs with stealth properties, we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that pluronics remained at the NP surface after dialysis (at one amphiphile per 5.5 nm2) and prevented NPs from nonspecific interactions with serum proteins and surfactants. In primary cultured neurons, pluronics stabilized the NPs, preventing their prompt aggregation and binding to neurons. By increasing dye loading to 20 wt % and optimizing particle size, we obtained 74 nm NPs showing 150-fold higher single-particle brightness with two-photon excitation than commercial Nile Red-loaded FluoSpheres of 39 nm hydrodynamic diameter. The obtained ultrabright pluronic-coated NPs enabled direct single-particle tracking in vessels of mice brains by two-photon intravital microscopy for at least 1 h, whereas noncoated NPs were rapidly eliminated from the circulation. Following brain injury or neuroinflammation, which can open the blood–brain barrier, extravasation of NPs was successfully monitored. Moreover, we demonstrated tracking of individual NPs from meningeal vessels until their uptake by meningeal macrophages. Thus, single NPs can be tracked in animals in real time in vivo in different brain compartments and their dynamics visualized with subcellular resolution.

Fluorescent probes are commonly used in studying G protein-coupled receptors in living cells; however their application to the whole animal receptor imaging is still challenging. To address this problem, we report the design and the synthesis of the first near-infrared emitting fluorogenic dimer with environment-sensitive folding. Due to the formation of non-fluorescent H-aggregates in an aqueous medium, the near-infrared fluorogenic dimer displays a strong turn-on response (up to 140-fold) in an apolar environment and exceptional brightness: 56% quantum yield and ≈444 000 M−1 cm−1 extinction coefficient. Grafted on a ligand of the oxytocin receptor, it allows the unprecedented background-free and target-specific imaging of the naturally expressed receptor in living mice.

Nanoemulsions (NEs) are biocompatible and stealth nanodroplets that can efficiently encapsulate hydrophobic cytoactive drugs in their oily core. NEs were shown to accumulate in tumors by enhanced permeability and retention (EPR) effect and thus display appealing features as nanocarriers to selectively deliver drugs to the tumors. However, to ensure efficient encapsulation with minimal early release, drugs must possess a high degree of lipophilicity. To circumvent this limitation, the latter could be transformed into prodrugs with enhanced hydrophobicity. In return, once delivered in the cell, the prodrug must be efficiently transformed into its active drug form. Herein we chemically and reversibly modified a near infrared Huda dye (HD) into pro-fluorophore (Pro-HD), a non-fluorescent and lipophilic prodrug model that was efficiently loaded in NEs. Thanks to the fluorogenecity of the system (fluorescence enhancement of 35-fold at 723 nm), we demonstrated that Pro-HD did not leak out of NEs, was efficiently delivered into cancer cells and was transformed in cellulo into HD. This proof of concept demonstrates the high potential of lipophilic "pro-fluorophore" approach for visualizing delivery of cargos using NEs as nanocarriers.

17 Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. 18 Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this 19 limitation, but they still suffer from poor brightness and photostability. Here, we propose a concept of 20 cell-permeable fluorogenic dimer of sulforhodamine B dyes (Gemini-561) and corresponding dimerized 21 aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The 22 unprecedented brightness and photostability together with high affinity of this complex allowed, for the 23 first time, direct fluorescence imaging in live mammalian cells of RNA polymerase-III transcription 24

Delivery systems able to coencapsulate both hydrophilic and hydrophobic species are of great interest in both fundamental research and industrial applications. Water-inoil-in-water (w 1 /O/W 2) emulsions are interesting systems for this purpose, but they suffer from limited stability. In this study, we propose an innovative approach to stabilize double emulsions by the synthesis of a silica membrane at the water/oil interface of the primary emulsion (i.e., inner w 1 /O emulsion). This approach allows the formulation of stable double emulsions through a twostep process, enabling high encapsulation efficiencies of model hydrophilic dyes encapsulated in the internal droplets. This approach also decreases the scale of the double droplets up to the nanoscale, which is not possible without silica stabilization. Different formulation and processing parameters were explored in order to optimize the methodology. Physicochemical characterization was performed by dynamic light scattering, encapsulation efficiency measurements, release profiles, and optical and transmission electron microscopies.

Uncontrolled release of encapsulated drugs and contrast agents from biodegradable polymer nanoparticles (NPs) is a central problem in drug delivery and bioimaging. In particular, it concerns polymeric NPs prepared by nanoprecipitation, where this release (so-called burst release) can be very significant, leading to side effects and/or bioimaging artifacts. Here, we made a systematic study on the effect of chemical structure of cargo molecules, BODIPY dye derivatives, on their capacity to be loaded into ~50 nm PLGA NPs without leakage in biological media. Absorption and fluorescence spectroscopy suggested that most of dyes, except the most polar BODIPY derivative, formed blended structures with polymer NPs. Fluorescence correlation spectroscopy of dye-loaded NPs in the presence of serum proteins revealed that only the most hydrophobic BODIPY dyes, bearing one octadecyl chain or two octyl chains, remain inside NPs, while all other derivatives are released into serum medium. The time-laps absorption and fluorescence studies confirmed this result, suggesting the release kinetics for the leaky NPs on the time scale of hours. Fluorescence microscopy of living cells incubated with BODIPY-loaded NPs showed that most of them exhibit strong dye leakage observed as homogeneous distribution of fluorescence all over the cytoplasm. Importantly, NPs loaded with the most hydrophobic dyes, exhibited high stability showing a dotted pattern in the perinuclear region, typical for endosomes and lysosomes. Our results highlight significance of the cargo hydrophobicity for efficient encapsulation inside polymeric NPs prepared by nanoprecipitation, which enables designing stable cargo-loaded nanomaterials for bioimaging and drug delivery. 2

In general, nano-emulsions are submicron droplets composed of liquid oil phase dispersed in liquid aqueous bulk phase. They are stable and very powerful systems when it regards the encapsulation of lipophilic compounds and their dispersion in aqueous medium. On the other hand, when the properties of the nano-emulsions aim to be modified, e.g. for changing their surface properties, decorating the droplets with targeting ligands, or modifying the surface charge, the dynamic liquid / liquid interfaces make it relatively challenging. In this study, we have explored the development of nano-emulsions which were not anymore stabilized with a classical low-molecular weight surfactant, but instead, with an amphiphilic polymer based on poly(maleic anhydride-alt-1-octadecene) (PMAO) and Jeffamine®, a hydrophilic amino-terminated PPG/PEG copolymer. Using a polymer as stabilizer is a potential solution for the nano-emulsion functionalization, ensuring the droplet stabilization as well as being a platform for the droplet decoration with ligands (for instance after addition of function groups in the terminations of the chains). The main idea of the present work was to understand if the spontaneous emulsification –commonly performed with nonionic surfactants– can be transposed with amphiphilic polymers, and a secondary objective was to identify the main parameters impacting on the process. PMAO was modified with two different Jeffamine®, additionally different oils and different formulation conditions were evaluated. As a control, the parent monomer, octadecyl succinic anhydride (OSA) was also modified and studied in the similar way as that of polymer. The generated nano-emulsions were mainly studied by dynamic light scattering and electron microscopy, that allows discriminating the crucial parameters in the spontaneous process, originally conducted with polymers as only stabilizer.

Staining of the plasma membrane (PM) is essential in bioimaging, as it delimits the cell surface and provides various information regarding the cell morphology and status. Herein, the lipophilicity of a green emitting BODIPY fluorophore was tuned by gradual functionalization with anchors composed of zwitterionic and aliphatic groups, thus yielding three different amphiphilic dyes. We found that BODIPY bearing one or three anchors failed in efficiently staining the PM: the derivative with one anchor showed low affinity to PM and exhibited strong fluorescence in water due to high solubility, whereas BODIPY with three anchors aggregated strongly in media and precipitated before binding to the PM. In sharp contrast, the BODIPY bearing two anchors (B-2AZ, MemBright-488) formed virtually nonfluorescent soluble aggregates in aqueous medium that quickly deaggregated in the presence of PM, leading to a bright soluble molecular form (quantum yield of 0.92). This fluorogenic response allowed for efficient probing of the PM at low concentration (20 nM) with high signal to background ratio images in mono- as well as two-photon excitation microscopy. B-2AZ proved to selectively stain the PM in a more homogeneous manner than the commercially available fluorescently labeled lectin WGA. Finally, it was successfully used in 3D-imaging to reveal fine intercellular tunneling nanotubes in KB cells and to stain the PM in glioblastoma cells in spheroids.

Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs’ hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo.

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M

Polymeric nanoparticles (PNPs) are gaining increasing importance as nanocarriers or contrasting material for preclinical diagnosis by micro-CT scanner. Here, we investigated a straightforward approach to produce a biocompatible, radiopaque, and stable polymer-based nanoparticle contrast agent, which was evaluated on mice. To this end, we used a nanoprecipitation dropping technique to obtain PEGylated PNPs from a preformed iodinated homopolymer, poly(MAOTIB), synthesized by radical polymerization of 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) monomer (MAOTIB). The process developed allows an accurate control of the nanoparticle properties (mean size can range from 140 nm to 200 nm, tuned according to the formulation parameters) along with unprecedented important X-ray attenuation properties (concentration of iodine around 59 mg I/mL) compatible with a follow-up in vivo study. Routine characterizations such as FTIR, DSC, GPC, TGA, 1H and 13C NMR, and finally SEM were accomplished to obtain the main properties of the optimal contrast agent. Owing to excellent colloidal stability against physiological conditions evaluated in the presence of fetal bovine serum, the selected PNPs suspension was administered to mice. Monitoring and quantification by micro-CT showed that iodinated PNPs are endowed strong X-ray attenuation capacity toward blood pool and underwent a rapid and passive accumulation in the liver and spleen.

Lipid droplets (LDs) are organelles that serve as the storage of intracellular neutral lipids. LDs regulate many physiological processes. They recently attracted attention after extensive studies showed their involvement in metabolic disorders and diseases such as obesity, diabetes, and cancer. Therefore, it is of the highest importance to have reliable imaging tools. In this review, we focus on recent advances in the development of selective fluorescent probes for LDs. Their photophysical properties are described, and their advantages and drawbacks in fluorescence imaging are discussed. At last, we review the reported applications using these probes including two-photon excitation, in vivo and tissue imaging, as well as LDs tracking

Ca-NIR is the first ratiometric fluorescent calcium probe emitting in the near infrared. , Fluorescent calcium probes are essential tools for studying the fluctuation of calcium ions in cells. Herein, we developed Ca-NIR, the first ratiometric calcium probe emitting in the near infrared region. This probe arose from the fusion of a BAPTA chelator and a dihydroxanthene-hemicyanine fluorophore. It is efficiently excited with common 630–640 nm lasers and displays two distinct emission bands depending on the calcium concentration ( K d = ∼8 μM). The physicochemical and spectroscopic properties of Ca-NIR allowed for ratiometric imaging of calcium distribution in live cells.

The rational design of environmentally sensitive dyes with superior properties is critical for elucidating the fundamental biological processes and understanding the biophysical behavior of cell membranes. In this study, a novel group of fluorene-based push-pull probes was developed for imaging membrane lipids. The design of these fluorogenic conjugates is based on a propioloyl linker to preserve the required spectroscopic features of the core dye. This versatile linker allowed the introduction of a polar deoxyribosyl head, a lipophilic chain, and an amphiphilic/anchoring group to tune the cell membrane binding and internalization. It was found that the deoxyribosyl head favored cell internalization and staining of intracellular membranes, whereas an amphiphilic anchor group ensured specific plasma membrane staining. The optimized fluorene probes presented a set of improvements as compared to commonly used environmentally sensitive membrane probe Laurdan such as red-shifted absorption matching the 405 nm diode laser excitation, a blue-green emission range complementary to the red fluorescent proteins, enhanced brightness and photostability, as well as preserved sensitivity to lipid order, as shown in model membranes and living cells.

Energy transfer efficiencies of UCNP-based RET systems are quantified through comparison of spectra, decay lifetimes, and semiempirical simulations.

Fluorescence detection sensitivity can be drastically improved by the application of nanoparticles (NPs)because of their superior brightness compared to organic dyes. Here, using dye-doped silica NPs(SiNPs), we developed FRET-based nanoparticle probes for the detection of reductive environments inliving cells. To this end, we designed three FRET acceptors based on black hole quenchers (BHQs). Theirpolarity was tuned by introducing hydroxyl, PEG and sulfate groups. To conjugate them to NPs, we usedan original pre-functionalization approach, where the quencher was coupled by a“click”reaction toPluronic F127 and further used for the preparation of silica NPs. This approach enabled easy preparationof silica NPs functionalized with varying amounts of quenchers by simple mixing of functionalized andparent Pluronic F127 in different mol%. The increase in the quencher concentration at the SiNPs surfaceproduced a rapid drop in thefluorescence intensity with 80% quenching and a 2-fold drop in theemission lifetime for 16 mol% of the quenchers. Then, to obtain turn-ON sensing of reductiveenvironments, the quenchers were coupled to the NPs through a disulfide linker using the same pre-functionalization strategy. The obtained nano-probes showed a >10-fold increase in theirfluorescencein the presence of reductive agents, such as tris(2-carboxyethyl)phosphine (TCEP) and glutathione.Remarkably, BHQ quencher bearing sulfate group showed the highest turn-ON response, probably dueto its superior capacity to escape from the NP surface after disulfide bond cleavage. The obtained bestnanoprobe was successfully applied for detection of reductive environments inside living cells usingfluorescence lifetime imaging (FLIM). This work provides insights for FRET acceptor design and itscontrolled grafting, which enables preparation of thefirst redox-sensitive silica nanoparticle probe forlifetime imaging.

Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The widespread development of fluorescent imaging techniques makes pH sensitive fluorescent dyes valuable tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine fluorophore that display a bright red fluorescence upon acidification with pK a values spanning from 4 to 9. Slight structural modifications led to dramatic changes in their physicochemical properties and a relationship between their structures, their ability to form H-aggregates, and their apparent pK a was established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) using flow cytometry.

The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca2+) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca2+ signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca2+ sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca2+ increases in astrocyte processes but not in somata. These Ca2+ increases systematically precede the onset of functional hyperemia by 1-2 s, reestablishing astrocytes as potential regulators of neurovascular coupling.

Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca 2+ and H + transients. Our sensors combine a commercially available CANdot ® 565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca 2+ or H + probes. These 'Rubies' are based on an extended rhodamine as a OPEN ACCESS Sensors 2015, 15 24663 fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H + or Ca 2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca 2+ and pH transients using live-cell fluorescence imaging.

A rational design of squaraine dyes with lipophilic and zwitterionic groups tunes cell entry, allowing for selective far-red/near-infrared imaging of plasma membrane vs. endoplasmic reticulum. They exhibit up to 110-fold fluorescence enhancement in biomembranes and enable cellular imaging at 1 nM concentration, which make them the brightest membrane probes to date.

Herein we developed a functionalizable OFF-ON red emitting fluorescent calcium probe based on a new chelating system formed by CuAAC click chemistry (Huisgen cycloaddition). The pro-sensor 7 which is not sensitive to Ca2+, contains an alkyne moiety that, upon the click reaction, forms a chelating group involving the 1,4-triazole. Probe 10 exhibited good sensitivity towards calcium (K-d = 5.8 mu M) and zinc (5.6 mu M) with a high dynamic range (65 fold fluorescence increase), high quantum yield (0.59) and showed very low fluorescence enhancement in the presence of a high concentration of Mg2+. We extended this method and generated two dextran conjugates in order to compare their sensing properties with those of the molecular form of 10.

Polarity-sensitive fluorogenic dyes raised considerable attention because they can turn on their fluorescence after binding to biological targets, allowing background-free imaging. However, their brightness is limited, and they do not operate in the far-red region. Here, we present a new concept of fluorogenic dye based on a squaraine dimer that unfolds on changing environment from aqueous to organic and thus turns on its fluorescence. In aqueous media, all three newly synthesized dimers displayed a short wavelength band characteristic of an H-aggregate that was practically nonfluorescent, whereas in organic media, they displayed a strong fluorescence similar to that of the squaraine monomer. For the best dimer, which contained a pegylated squaraine core, we obtained a very high turn-on response (organic vs aqueous) up to 82-fold. Time-resolved studies confirmed the presence of nonfluorescent intramolecular H-aggregates that increased with the water content. To apply these fluorogenic dimers for targeted imaging, we grafted them to carbetocin, a ligand of the oxytocin G protein-coupled receptor. A strong receptor-specific signal was observed for all three conjugates at nanomolar concentrations. The probe derived from the core-pegylated squaraine showed the highest specificity to the target receptor together with minimal nonspecific binding to serum and lipid membranes. The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having similar to 660,000 M(-)1 cm(-)1 extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications. The proposed concept can be extended to other dye families and other membrane receptors, opening the route to new ultrabright fluorogenic dyes.

The key to ultrabright fluorescent nanomaterials is the control of dye emission in the aggregated state. Here, lipophilic rhodamine B derivatives are assembled into nanoparticles (NPs) using tetraphenylborate counterions with varied fluorination levels that should tune the short-range dye ordering. Counterion fluorination is found to drastically enhance the emission characteristics of these NPs. Highly fluorinated counterions produce 10-20 nm NPs containing >300 rhodamine dyes with a fluorescence quantum yield of 40-60% and a remarkably narrow emission band (34 nm), whereas, for other counterions, aggregation caused quenching with a weak broad-band emission is observed. NPs with the most fluorinated counterion (48 fluorines) are ∼40-fold brighter than quantum dots (QD585 at 532 nm excitation) in single-molecule microscopy, showing improved photostability and suppressed blinking. Due to exciton diffusion, revealed by fluorescence anisotropy, these NPs are efficient FRET donors to single cyanine-5 acceptors with a light-harvesting antenna effect reaching 200. Finally, NPs with the most fluorinated counterion are rather stable after entry into living cells, in contrast to their less fluorinated analogue. Thus, the present work shows the crucial role of counterion fluorination in achieving high fluorescence brightness and photostability, narrow-band emission, efficient energy transfer and high intracellular stability of nanomaterials for light harvesting and bioimaging applications.

: Small-molecule chemical calcium (Ca2+) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca2+ entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as a promising alternative, but their use to monitor intracellular processes is still a major challenge. We designed cell-penetrating FRET-based Ca2+ nanobiosensors for the detection of local Ca2+ concentration transients, using commercially available CANdot®565QD as a donor and CaRuby, a custom red-emitting Ca2+ indicator as an acceptor. With Ca2+ -binding affinity in the range of 3 to 20 µM, our CaRubies allow sensors to be scalable for detecting intracellular Ca2+ transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-D-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca2+concentration changes following agonist application as evidenced by TIRF imaging. Our work presents the synthesis of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca2+ transients.

: Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and on core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, have intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalogue of criteria for evaluating Ca(2+) indicators that should be made available for each probe. This invited review article is part of the special issue 'Calcium signaling as a hub for translational medicine and a starting point to model life'. (275 words).

Herein is described the synthesis and spectroscopic characterizations of three new OFF ON red-emitting and water-soluble sensors, CAXR (Clicked APTRA X-Rhodamine). These dyes are based on an extended APTRA (aminophenol triacetic acid) motif. Three different side chains were added by click chemistry in order to complete the coordination sphere with a chelate moiety composed of a triazolyl and an iminol. The fluorescent response (F/F-0) of these probes follows the order: Cd2+>Zn2+>Pb2+>Hg2+. An important and unexpected effect of the side chain structure on the Kd was observed (up to one order of magnitude, Cadmium Kd from 252 to 21 mu M). This remote effect of the side chains was studied by DFT calculations and was attributed to a twisted conformation of the CAXR-Py:Cd2+ complex.

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.

Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca2+) indicators, were designed and synthesized as new tools for intracellular Ca2+ imaging. The attachment of a side arm on the ethylene glycol bridge makes it possible to link the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca2+ binding affinity. Using this approach it has been possible to characterize three different CaRubies having affinities between 3 and 22 µM. Using click chemistry, we demonstrate high-yield linkage of the azido form of their arm to PEG molecules that can be used, e.g. the stoichiometric design of ratiometric, FRET-based indicators. The long excitation and emission wavelengths of CaRubies allow otherwise challenging multi-color experiments, e.g., when combining Ca2+ uncaging or optogenetic stimulation with Ca2+ imaging. We illustrate this capacity by the detection with CaRubies of blue-light-evoked Ca2+ transients in cultured astrocytes expressing CatCh, a light-sensitive Ca2+-translocating channelrhodopsin, linked to yellow fluorescent protein for the identification of transfected cells. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and show a roughly tenfold increase in the average lifetime upon Ca2+ chelation. Since only the fluorescence quantum yield of the CaRubies is Ca2+-dependent, calibrated measurements of absolute Ca2+ concentrations are possible with single-wavelength two-photon fluorescence excitation.

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.

Amphiphilic cyclodextrins (CDs) are good candidates to functionalize natural membranes as well as synthetic vesicles. In this paper, we provide a full description of the interfacial behavior of pure 6I,6IV-(β-cholesteryl)succinylamido-6I,6IV-(6-deoxy-per-(2,3,6-O-methyl))cycloheptaose (TBdSC) and how it inserts in dipalmitoyl-l-α-phosphatidylcholine (DPPC) monolayers as a membrane model. Langmuir isotherms of pure TBdSC suggest a reorganization upon compression, which could be clarified using X-ray reflectivity. The CD head can adjust its conformation to the available area per molecule. A compatible model involving a rotation around a horizontal axis defined by the two selectively substituted glucose units is proposed. The in-plane structure is characterized at all scales by Brewster angle microscopy (BAM) on the water surface and atomic force microscopy (AFM) on monolayers deposited on solid substrates. The same tools are used for its mixtures with DPPC. We show in particular that TBdSC seems to be soluble in the liquid-expanded DPPC. However, phase segregation occurs at higher pressure, allowing for sequentially liquid-condensed DPPC and high-pressure conformation of TBdSC. This gives rise to a remarkable contrast inversion in both imaging methods.

Four biotinylated tri and tetrasaccharide fragments of plant and invertebrate N-glycans were synthesized using methyl tert-butyl phenyl (MBP) thioglycosides donors in order to evaluate their involvement in cross-allergies as cross-reactive carbohydrate determinants (CCDs). Various levels of reactivity to anti-bee and anti-HRP antibodies and with sera from allergic patients were observed when the conjugates were coated on streptavidin microplates. The results showed the potential utility of these xylosylated and fucosylated oligosaccharide fragments in determining CCD antibody epitopes.

The synthesis of a tetra β(1→5) galactofuranoside was achieved using a thioglycoside donor with a methyl tert-butyl phenyl thio leaving group. This tetrasaccharide was conjugated to biotin and validated as antigen with the monoclonal antibody used for clinical detection of Aspergillus fumigatus galactomannan on streptavidin-coated microplates. Then we have shown its ability to detect antibodies associated with A. fumigatus induced disease by using sera from patients with Allergic broncho-pulmonary aspergillosis (ABPA) and correlated the results of antibody detection with those gained with a commercially available diagnostic test.

As a part of our glycoantigen synthetic program for diagnosis and basic analysis of yeast-related pathogenic mechanisms, a library of 1-->2 oligomannosides suitable for immunoanalysis was prepared. The use of biotin sulfone, an oxidized form of biotin, offers a convenient solution for both oligosaccharide synthesis and immobilization on microspheres and surface plasmon resonance sensors. The application of this new strategy for the analysis of anti- Candida albicans antibody response through multiple-analyte profiling technology (Luminex) and with surface plasmonic analysis using biotin tagged synthetic oligosaccharides on avidin coated surfaces was validated using monoclonal antibodies.

OBJECTIVES: Anti-S. cerevisiae mannan antibodies (ASCA) are human antibodies associated with Crohn's disease (CD) reacting with Saccharomyces cerevisiae (S. cerevisiae) mannan polymer. As mannan is a complex and variable repertoire of oligomannoses acting as epitopes, we chemically synthesized (Sigma) two major oligomannose epitopes, Man alpha-1,3 Man alpha-1,2 Man (SigmaMan3) and Man alpha-1,3 Man alpha-1,2 Man alpha-1,2 Man (SigmaMan4), and then explored how antisynthetic mannoside antibodies (ASigmaMA) compare with ASCA as markers of CD. METHODS: The study involved different cohorts of CD and ulcerative colitis (UC) patients and healthy controls who had been studied previously in several medical centers in Europe, the United States, and North Africa to determine the clinical value of ASCA in terms of differential diagnosis, evolution of indeterminate colitis (IC), and serotype-phenotype correlations. The comparison of ASigmaMA and ASCA included a total of 1,365 subjects: 772 CD, 261 UC, 43 IC, and 289 controls. RESULTS: The specificity of ASigmaMA was similar to that of ASCA (89% vs 93%), although the sensitivity was lower (38% vs 55%). Unexpectedly, 24% of the CD patients who were negative for ASCA and/or other CD-associated serologic markers were positive for ASigmaMA. ASigmaMA were associated with colonic involvement in CD (odds ratio [OR] 1.609, 95% confidence interval [CI] 1.033-2.506, P = 0.03) and were 100% predictive of CD in patients with IC. CONCLUSIONS: ASigmaMA reveal the heterogeneity of the antioligomannose antibody response in CD patients and increase the sensitivity of CD diagnosis when combined with ASCA. The subset of ASCA-negative CD patients diagnosed by ASigmaMA had preferentially a colonic involvement, which confirms the high predictive value of ASigmaMA for determining IC evolution toward CD.

<div>Hypothesis<p>Characterization of nanoscale formulations is a continuous challenge. Size, morphology and surface properties are the most common characterizations. However, physicochemical properties inside the nanoparticles, like viscosity, cannot be directly measured. Herein, we propose an original approach to measuring dynamic viscosity using a lipidic molecular rotor solubilized in the core of nano-formulations. These molecules undergo conformational changes in response to viscosity variations, leading to observable changes in fluorescence intensity and lifetime, able to sense the volume properties of dispersed nano-domains.</p></div> <div>Experiments<p>The lipophilic molecular rotor (BOPIDY derivatives) was specifically synthesized and characterized as oil viscosity sensing in large volumes. A second part of the study compares these results with rBDP-Toco in nano-emulsions. The objective is to evaluate the impact of the formulation, droplet size and composition on the viscosity of the droplet's core.</p></div> <div>Findings<p>The lipophilic rotor showed a universal behavior whatever the oil composition, giving a master curve. Applied to nano-formulations, it discloses the viscosity in the nano-emulsion droplets, enabling the detection of slight variations between reference oil samples and the nanoformulated ones. This new tool opens the way to the fine characterization of complex colloids and multi-domain nano and micro systems, potentially applied to hybrid materials and biomaterials.</p></div>