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–1 cm–1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.
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.
Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role ...in a variety of physiological processes as well as diseases. Herein we synthesized a push–pull molecule named DAF (Dimethyl Aniline Furaldehyde) that possesses a strong positive solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410–500 nm) and cytoplasm in a red channel (500–600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.
The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright, a family of six cyanine-based fluorescent turn-on PM probes that ...emit from orange to near infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono- and two-photon microscopy. These probes are compatible with long-term live-cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells, allowing identification of neurons in acute brain tissue sections and neuromuscular junctions without any use of transfection or transgenic animals. In addition, MemBright probes were used in super-resolution imaging to unravel the neck of dendritic spines. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques. VIDEO ABSTRACT.
Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalent in the extracellular matrix. The synthesis of structurally defined GAGs requires laborious procedures, and incorporating ...defined sulfation patterns is challenging. The automated synthesis of defined sulfated chondroitin hexasaccharides on solid support has been achieved using a photolabile linker that is efficiently cleaved in a continuous‐flow photoreactor.
Lipid nanoemulsions (NEs), owing to their controllable size (20 to 500 nm), stability and biocompatibility, are now frequently used in various fields, such as food, cosmetics, pharmaceuticals, drug ...delivery, and even as nanoreactors 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, the composition of dye-loaded NEs, methods for their preparation, and emerging biological applications are described. The design of bright fluorescent NEs with high dye loading and minimal aggregation-caused quenching (ACQ) is focused on. 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. Thereafter, biological applications for bioimaging and single-particle tracking in cells and small animals as well as biomedical applications for photodynamic therapy are described. Finally, challenges and future perspectives of fluorescent NEs are discussed.
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.
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•MemBright allows for bright and specific staining of EVs•The zebrafish embryo allows tracking of tumor EVs at high spatiotemporal resolution•Circulating tumor EVs are mostly taken up by endothelial cells and patrolling macrophages•Zebrafish melanoma EVs favor metastatic outgrowth in zebrafish embryos
Tumor extracellular vesicles (EVs) promote tumor progression. However, their behavior in body fluids remains mysterious. Hyenne et al. show that the zebrafish embryo can be used to track and assess the function of circulating tumor EVs in vivo and provide a high-resolution description of their dissemination and uptake.
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 cross-talk, higher brightness, improved photostability) and, unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell types including neurons (primary culture and tissue imaging). Taking advantage of these features, we successfully used SP-468 in STED super resolution neuronal imaging. Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we showed that the new probes kept the ability 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.
Abstract
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.
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.
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