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Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.
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Barrera Hematoencefálica , Tomografía con Microscopio Electrónico , Animales , Ratones , Tomografía con Microscopio Electrónico/métodos , Barrera Hematoencefálica/ultraestructura , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/ultraestructura , Ratones Endogámicos C57BL , Masculino , Microscopía Electrónica de Rastreo/métodos , MicrotomíaRESUMEN
Imaging and sensing of lipid droplets (LDs) attracted significant attention due to growing evidence for their important role in cell life. Solvatochromic dyes are promising tools to probe LDs' local polarity, but this analysis is biased by their non-negligible emission from intracellular membranes and capacity to emit from both the apolar core and polar interface of LDs. Here, we developed two push-pull solvatochromic dyes based on naphthalene and fluorene cores bearing an exceptionally strong electron acceptor, the trifluoroacetyl group. The latter was found to boost the optical properties of the dyes by shifting their absorption and emission to red and increasing their extinction coefficient, photostability, and sensitivity to solvent polarity (solvatochromism). In contrast to classical solvatochromic dyes, such as parent aldehydes and reference Nile Red, the new dyes exhibited strong fluorescence quenching by millimolar water concentrations in organic solvents. In live cells, the trifluoroacetyl dyes exhibited high specificity to LDs, whereas the parent aldehydes and Nile Red showed a detectable backgrounds from intracellular membranes. Experiments in model lipid membranes and nanoemulsion droplets confirmed the high selectivity of new probes to LDs in contrast to classical solvatochromic dyes. Moreover, the new probes were found to be selective to the LDs oil core, where they can sense lipid unsaturation and chain length. Their ratiometric imaging in cells revealed strong heterogeneity in polarity within LDs, which covered the range of polarities of unsaturated triglyceride oils, whereas Nile Red failed to properly estimate the local polarity of LDs. Finally, the probes revealed that LDs core polarity can be altered by fatty acid diets, which correlates with their chain length and unsaturation.
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Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10â 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362 nt with the limit of detection down to 0.3 fm using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.
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Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VHHs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VHHs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.
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Nanopartículas , Péptidos , Nanopartículas/química , Animales , Humanos , Ratones , Péptidos/química , Línea Celular Tumoral , Femenino , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Neoplasias de la Mama/metabolismoRESUMEN
Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment-sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.
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Colorantes Fluorescentes , Protones , Colorantes Fluorescentes/química , Proteínas , Aminoácidos , LípidosRESUMEN
The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.
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Cuerpo Celular , Conos de Crecimiento , Neuronas/metabolismo , Membrana Celular , Hipocampo , Células CultivadasRESUMEN
Polymer nanoparticles (NPs) loaded with drugs and contrast agents have become key tools in the advancement of nanomedicine, requiring robust technologies for their synthesis. Nanoprecipitation is a particularly interesting technique for the assembly of loaded polymer NPs, which is well-known to proceed under kinetic control, with a strong influence of the assembly conditions. On the other hand, the nature of the used polymer also influences the outcome of nanoprecipitation. Here, we investigated systematically the relative effects of mixing of the organic and aqueous phases and polymer chemistry on the formation of polymer nanocarriers. For this, two mixing schemes, manual mixing and microfluidic mixing using an impact-jet micromixer, were first evaluated, showing mixing times of several tens of milliseconds and a few milliseconds, respectively. Copolymers of ethyl methacrylate with charged and hydrophilic groups and different polyesters (poly(d-l-lactide-co-glycolide) and poly(lactic acid)) were combined with a fluorescent dye salt and tested for particle assembly using these "slow" and "fast" mixing methods. Our results showed that in the case of the most hydrophobic polymers, the speed of mixing had no significant influence on the size and loading of the formed NPs. In contrast, in the case of less hydrophobic polymers, faster mixing led to smaller NPs with better encapsulation. The switch between mixing and polymer-controlled assembly was directly correlated to the solubility limit of the polymers in acetonitrile-water mixtures, with a critical point for solubility limits between 15 and 20 vol % of water. Our results provide simple guidelines on how to evaluate the possible influence of polymer chemistry and mixing on the formation of loaded NPs, opening the way to fine-tune their properties and optimize their large-scale production.
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The aggregation in a solution of charged dyes such as Rhodamine B (RB) is significantly affected by the type of counterion, which can determine the self-assembled structure that in turn modulates the optical properties. RB aggregation can be boosted by hydrophobic and bulky fluorinated tetraphenylborate counterions, such as F5TPB, with the formation of nanoparticles whose fluorescence quantum yield (FQY) is affected by the degree of fluorination. Here, we developed a classical force field (FF) based on the standard generalized Amber parameters that allows modeling the self-assembling process of RB/F5TPB systems in water, consistent with experimental evidence. Namely, the classical MD simulations employing the re-parametrized FF reproduce the formation of nanoparticles in the RB/F5TPB system, while in the presence of iodide counterions, only RB dimeric species can be formed. Within the large, self-assembled RB/F5TPB aggregates, the occurrence of an H-type RB-RB dimer can be observed, a species that is expected to quench RB fluorescence, in agreement with the experimental data of FQY. The outcome provides atomistic details on the role of the bulky F5TPB counterion as a spacer, with the developed classical FF representing a step towards reliable modeling of dye aggregation in RB-based materials.
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Brightness is a fundamental property of fluorescent nanomaterials reflecting their capacity to absorb and emit light. In sensing materials, brightness is crucial for high-sensitivity (bio)molecular detection, while in optical bioimaging it ensures high spatial and temporal resolution. Fluorescent organic nanoparticles (NPs) are particularly attractive because of their superior brightness compared to organic dyes. With the ever-growing diversity of organic nanomaterials, it is important to establish universal principles for measuring and estimating their brightness. This tutorial review provides definitions of brightness and describes the major approaches to its analysis based on ensemble and single-particle techniques. We present the current chemical approaches to fight Aggregation-Caused Quenching (ACQ) of fluorophores, which is a major challenge in the design of bright organic nanomaterials. The main classes of fluorescent organic NPs are described, including conjugated polymer NPs, aggregation-induced emission NPs, and NPs based on neutral and ionic dyes. Their brightness and other properties are systematically compared. Some brightest examples of bulk solid-state emissive organic materials are also mentioned. Finally, we analyse the importance of brightness and other particle properties in biological applications, such as bioimaging and biosensing. This tutorial will provide guidelines for chemists on the design of fluorescent organic NPs with improved performance and help them to estimate and compare the brightness of new nanomaterials with literature reports. Moreover, it will help biologists to select appropriate materials for sensing and imaging applications.
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A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.
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Colorantes Fluorescentes , Orgánulos , Colorantes Fluorescentes/química , Orgánulos/química , Membrana Celular/metabolismo , Aparato de Golgi , Retículo Endoplásmico , Proteínas de la Membrana/metabolismoRESUMEN
Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics, and biosensing, but its operating range is fundamentally limited by the Förster radius of ≈5 nm. In this work, FRET between fluorescent organic nanoparticles (NPs) is studied in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distances of 15 and 20 nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this long-distance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding the cancer marker survivin, bringing together donor and acceptor NPs at ≈15 nm distance. In this nanoprobe, a single-molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based biosensing.
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Nanopartículas , Transferencia Resonante de Energía de Fluorescencia , Nanopartículas/química , ADN/química , Colorantes Fluorescentes/químicaRESUMEN
Invited for the cover of this issue is the group of Mayeul Collot at the University of Strasbourg (CNRS). The image depicts the effect of simple chemical tuning on coumarin dyes to tune and improve the DPIC photoconversion mechanism. Read the full text of the article at 10.1002/chem.202203933.
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The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers.
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Colorantes Fluorescentes , Nanopartículas , Colorantes Fluorescentes/química , Biotina/química , Biotina/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Cetuximab , Nanopartículas/química , Polímeros/químicaRESUMEN
Dual-emissive photoconvertible fluorophores (DPCFs) are powerful tools to unambiguously track labeled cells in bioimaging. We recently introduced a new rational mechanism called directed photooxidation-induced conversion (DPIC) enabling efficient DPCFs to be obtained by conjugating a coumarin to aromatic singlet-oxygen reactive moieties (ASORMs). Pyrrole was found to be a suitable ASORM as it provided a high hypsochromic shift along with a fast and efficient conversion. By synthesizing various pyrrole-based styryl coumarin dyes, we showed that the photoconversion properties, including the quantum yield of photoconversion and the chemical yield of conversion can be tuned by chemical modification of the pyrrole. These modifications led to an improved dual emissive converter, SCP-Boc, which displayed a high brightness and an enhanced photoconversion yield of 63 %. SCP-Boc was successfully used to sequentially photoconvert cells by laser scanning confocal microscopy.
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Current biomedical applications of nanocarriers are focused on drug delivery, where encapsulated cargo is released in the target tissues under the control of external stimuli. Here, we propose a very different approach, where the active toxic molecules are removed from biological tissues by the nanocarrier. It is based on the drug-sponge concept, where specific molecules are captured by the lipid nanoemulsion (NE) droplets due to dynamic covalent chemistry inside their oil core. To this end, we designed a highly lipophilic amine (LipoAmine) capable of reacting with a free cargo-aldehyde (fluorescent dye and 4-hydroxynonenal toxin) directly inside lipid NEs, yielding a lipophilic imine conjugate well encapsulated in the oil core. The formation of imine bonds was first validated using a push-pull pyrene aldehyde dye, which changes its emission color during the reaction. The conjugate formation was independently confirmed by mass spectrometry. As a result, LipoAmine-loaded NEs spontaneously loaded cargo-aldehydes, yielding formulations stable against leakage at pH 7.4, which can further release the cargo in a low pH range (4-6) in solutions and living cells. Using fluorescence microscopy, we showed that LipoAmine NEs can extract pyrene aldehyde dye from cells as well as from an epithelial tissue (chicken skin). Moreover, successful extraction from cells was also achieved for a highly toxic aliphatic aldehyde 4-hydroxynonenal, which allowed obtaining the proof of concept for detoxification of living cells. Taken together, these results show that the dynamic imine chemistry inside NEs can be used to develop detoxification platforms.
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Portadores de Fármacos , Iminas , Portadores de Fármacos/química , Preparaciones de Acción Retardada , Aldehídos , LípidosRESUMEN
In this study, we explored how chemical reactions of amphiphile compounds can be characterized and followed-up on model interfaces. A custom-made surfactant containing three alkyne sites was first adsorbed and characterized at a water/oil interface. These amphiphiles then underwent interfacial crosslinking by click chemistry upon the addition of a second reactive agent. The monolayer properties and dilatational elasticity, were compared before and after the polymerization. Using bulk phase exchange, the composition of the aqueous bulk phase was finely controlled and washed to specifically measure the interfacial effects of the entities adsorbed and trapped at the interface. In this study, we aim to emphasize an original experimental approach to follow complex phenomena occurring on model interfaces, and also show the potential of this method to characterize multifactorial processes.
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Surfactantes Pulmonares , Tensoactivos , Tensoactivos/química , Agua/química , Química Clic , AdsorciónRESUMEN
Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. The development of chemical toolsâfluorescent probesâfor the sensing and imaging of biomembranes is a rapidly growing research direction, stimulated by a high demand from cell biologists and biophysicists. This Account focuses on advances in these smart molecules, providing a voyage from the cell frontierâplasma membranes (PM)âtoward intracellular membrane compartmentsâorganelles. General classification of the membrane probes can be based on targeting principles, sensing profile, and optical response. Probes for PM and organelle membranes are designed based on multiple targeting principles: conjugation with natural lipids or synthetic targeting ligands and in situ cell labeling by bio-orthogonal chemistry, conjugation to protein tags, and receptor-ligand interactions. Thus, to obtain membrane probes targeting PM with selectivity to one leaflet, we designed membrane anchor ligands based on a charged group and an alkyl chain. According to the sensing profile, we define basic membrane markers with constant emission and probes for biophysical and chemical sensing. The markers are built from classical fluorophores, exemplified by a series of bright cyanines and BODIPY dyes bearing the PM anchors (MemBright). Membrane probes for biophysical sensing are based on environment-sensitive fluorophores: (1) polarity-sensitive solvatochromic dyes; (2) viscosity-sensitive fluorescent molecular rotors; (3) mechanosensitive fluorescent flippers; and (4) voltage-sensitive electrochromic dyes. Our solvatochromic probes based on Nile Red (NR12S, NR12A, NR4A), Laurdan (Pro12A), and 3-hydroxyflavone (F2N12S) through polarity-sensing can visualize liquid ordered and disordered phases of lipid membranes, sense lipid order and its heterogeneity in cell PM, detect apoptosis, etc. Chemically sensitive probes, combining a dye, membrane-targeting ligand, and molecular recognition unit, enable the detection of pH, ions, redox species, lipids, and proteins at the biomembrane surface. In terms of the optical response profile, we can identify (1) fluorogenic (turn-on) probes, allowing background-free imaging; (2) ratiometric probes, e.g., solvatochromic probes, which enable ratiometric imaging by changing their emission/excitation color; (3) fluorescence lifetime-responsive probes, e.g., fluorescence molecular rotors and flippers, suitable for fluorescence lifetime imaging (FLIM); and (4) switchable probes, important for single-molecule localization microscopy. We showed that combining solvatochromic probes with on-off switching through a reversible binding specifically to cell PM enables the mapping of their biophysical properties with superior resolution. While the majority of efforts have been focused on PM, the probes for cellular organelles, such as endoplasmic reticulum, mitochondria, Golgi apparatus, etc., emerge rapidly. Thus, nontargeted solvatochromic probes can distinguish organelles by the emission color. Targeted solvatochromic probes based on Nile Red revealed unique signatures of polarity and lipid order of individual organelles and their different sensitivities to oxidative or mechanical stress. Lipid droplets, which are membraneless lipidic structures, constitute another interesting organelle target for probing the cell stress. Currently, we stand at the beginning of a long route with big challenges ahead, in particular (1) to achieve superior organelle specificity; (2) to label specific biomembrane leaflets, notably the inner leaflet of PM; (3) to detect lipid organization in a proximity of specific proteins; and (4) to probe biomembranes in tissues and animals.
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Colorantes Fluorescentes , Orgánulos , Animales , Colorantes Fluorescentes/química , Ligandos , Membrana Celular/metabolismo , Lípidos/químicaRESUMEN
Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.
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Nanopartículas del Metal , Nanopartículas , Oro/química , Nanopartículas/química , Polímeros , Especies Reactivas de Oxígeno/metabolismo , Nanopartículas del Metal/químicaRESUMEN
We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORMs) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68â nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.
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Colorantes Fluorescentes , Luz , Procesos Fotoquímicos , CumarinasRESUMEN
Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and â¼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.