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.
Dye-loaded lipid nano-droplets present an attractive alternative to inorganic nanoparticles, as they are composed of non-toxic biodegradable materials and easy to prepare. However, to achieve high ...fluorescence brightness, the nano-droplets have to be heavily loaded with the dyes avoiding fluorescence self-quenching and release (leakage) of the encapsulated dyes from the nano-droplets in biological media. In the present work, we have designed highly lipophilic fluorescent derivatives of 3-alkoxyflavone (F888) and Nile Red (NR668) that can be encapsulated in the lipophilic core of stable nano-emulsion droplets at exceptionally high concentrations in the oil core,
up to 170 mM and 17 mM, respectively, corresponding to ~ 830 and 80 dyes per 40-nm droplet. Despite this high loading, these dyes keep high fluorescence quantum yield and thus, provide high nano-droplet brightness, probably due to their bulky structure preventing self-quenching. Moreover, simultaneous encapsulation of both dyes at high concentrations in single nano-droplets allows observation of FRET. FRET and fluorescence correlation spectroscopy (FCS) studies showed that NR668 release in the serum-containing medium is very slow, while the reference hydrophobic dye Nile Red leaks immediately. This drastic difference in the leakage profile between NR668 and Nile Red was confirmed by
cellular studies as well as by
angiography imaging on zebrafish model, where the NR668-loaded nano-droplets remained in the blood circulation, while the parent Nile Red leaked rapidly from the droplets distributing all over the animal body. This study suggests new molecular design strategies for obtaining bright nano-droplets without dye leakage and their use as efficient and stable optical contrast agents
and
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In this study, we investigated the role of the chemical nature of the oil droplet core of nano-emulsions used as contrast agents for X-ray imaging on their pharmacokinetics and biodistribution. To ...this end, we formulated PEGylated nano-emulsions with two iodinated oils (i.e., iodinated monoglyceride and iodinated castor oil) and compared them with another iodinated nano-emulsion based on iodinated vitamin E. By using dynamic light scattering and transmission electron microscopy, the three iodinated nano-emulsions were found to exhibit comparable morphologies, size, and surface composition. Furthermore, they were shown to be endowed with very high iodine concentration, which leads to stronger X-ray attenuation properties as compared to the commercial iodinated nano-emulsion Fenestra VC. The three nano-emulsions were i.v. administered in mice and monitored by microcomputed tomography (micro-CT). They showed high contrast enhancement in blood with similar half-life around 6 h but very different accumulation sites. While iodinated monoglycerides exhibited low accumulation in liver and spleen, high accumulation in spleen was observed for iodinated castor oil and in liver for vitamin E. These data clearly highlighted the important role of the oil composition of the nano-emulsion core to obtain strong X-ray contrast enhancement in specific targets such as liver, spleen, or only blood. These differences in biodistribution were partly attributed to differences in the uptake of the nanodroplets by the macrophages in vitro. Another key feature of these nano-emulsions is their long half-elimination time (several weeks), which offers sufficient retention for micro-CT imaging. This work paves the way for the design of nanoparticulate contrast agents for X-ray imaging of selected organs.
During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early ...phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.
Nanoemulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly(ethylene glycol), ...through the use of nonionic surfactants, confers high stealth in biological medium with reduced nonspecific interactions. Tracking individual NE by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new dioxaborine barbituryl styryl (DBS) dyes that displayed high molar extinction coefficients (up to 120 000 M–1 cm–1) with relatively low quantum yields in solvents and impressive fluorescence enhancement when dissolved in viscous oils (up to 0.98). The reported screening of nine different oils allowed disclosing a range of efficient “oil/dye” couples and understanding the main parameters that lead to the brightest NEs. We determine vitamin E acetate/DBS-C8 as the representative most efficient couple, combining high dye loading capabilities and low aggregation-induced quenching, leading to <50 nm ultrabright NEs (with brightness as high as 30 × 106 M–1 cm–1) with negligible dye leakage in biological media. Beyond a comprehensive optical and physicochemical characterization of fluorescent NEs, cellular two-photon excitation imaging was performed with polymer-coated cell penetrating NEs. Thanks to their impressive brightness and photostability, NEs displaying different charge surfaces were microinjected in HeLa cells and were individually tracked in the cytosol to study their relative velocity.
The human immunodeficiency virus (HIV-1) polyprotein Gag (Group-specific antigen) plays a central role in controlling the late phase of the viral lifecycle. Considered to be only a scaffolding ...protein for a long time, the structural protein Gag plays determinate and specific roles in HIV-1 replication. Indeed, via its different domains, Gag orchestrates the specific encapsidation of the genomic RNA, drives the formation of the viral particle by its auto-assembly (multimerization), binds multiple viral proteins, and interacts with a large number of cellular proteins that are needed for its functions from its translation location to the plasma membrane, where newly formed virions are released. Here, we review the interactions between HIV-1 Gag and 66 cellular proteins. Notably, we describe the techniques used to evidence these interactions, the different domains of Gag involved, and the implications of these interactions in the HIV-1 replication cycle. In the final part, we focus on the interactions involving the highly conserved nucleocapsid (NC) domain of Gag and detail the functions of the NC interactants along the viral lifecycle.
Pulsatile flow is a universal feature of the blood circulatory system in vertebrates and can lead to diseases when abnormal. In the embryo, blood flow forces stimulate vessel remodeling and stem cell ...proliferation. At these early stages, when vessels lack muscle cells, the heart is valveless and the Reynolds number (Re) is low, few details are available regarding the mechanisms controlling pulses propagation in the developing vascular network. Making use of the recent advances in optical-tweezing flow probing approaches, fast imaging and elastic-network viscous flow modeling, we investigated the blood-flow mechanics in the zebrafish main artery and show how it modifies the heart pumping input to the network. The movement of blood cells in the embryonic artery suggests that elasticity of the network is an essential factor mediating the flow. Based on these observations, we propose a model for embryonic blood flow where arteries act like a capacitor in a way that reduces heart effort. These results demonstrate that biomechanics is key in controlling early flow propagation and argue that intravascular elasticity has a role in determining embryonic vascular function.
Graphene‐based 2D nanomaterials exhibit unique physicochemical, electric, and optical properties that facilitate applications in a wide range of fields including material science, electronics, and ...biotechnology. Recent studies have shown that graphene oxide (GO) and reduced graphene oxide (rGO) exhibit antimicrobial effects on bacteria and viruses. While the bactericidal activity of graphene‐based nanomaterials is related to mechanical and oxidative damage to bacterial membranes, their antiviral activity has been less explored. Currently available experimental data are limited and suggest mechanical disruption of viral particles prior to infection. In this study, the antiviral properties of reduced GO‐based nanocomposites decorated with Ag nanoparticles (rGO‐Ag) are evidenced against human immunodeficiency virus‐1 pseudovirus used as an enveloped virus model. By combining biochemical and original single virus imaging approaches, it is shown that rGO‐Ag induces peroxidation of pseudoviral lipid membrane and that consequent alteration of membrane properties leads to a reduction in cell entry. In addition, rGO‐Ag is found to be efficiently internalized in the host cell leading to the elevated expression of pro‐inflammatory cytokines. Altogether, the presented results shed new light on the mechanisms of rGO‐Ag antiviral properties and confirm the high potential of graphene derivatives as an antimicrobial material for biomedical applications.
Silver nanoparticle‐decorated reduced graphene oxide composites (rGO‐Ag) present antiviral activity against human immunodeficiency virus‐1‐based pseudovirus. The results show that rGO‐Ag induce peroxidation of the viral lipid envelope, perturb its biophysical properties, and impede viral entry into the host cell. In parallel, rGO‐Ag are internalized by cells and stimulate the transcription of pro‐inflammatory genes, which most probably contribute to infection inhibition.
The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 ...virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In most of the studies, nano-emulsion characterization is limited to their size distribution and zeta potential. In this review, we present an updated insight of the characterization methods of ...nano-emulsions, including new or unconventional experimental approaches to explore in depth the nano-emulsion properties.
We propose an overview of all the main techniques used to characterize nano-emulsions, including the most classical ones, up to in vitro, ex vivo and in vivo evaluation. Innovative approaches are then presented in the second part of the review that presents innovative, experimental techniques less known in the field of nano-emulsion such as the nanoparticle tracking analysis, small-angle X-ray scattering, Raman spectroscopy, and nuclear magnetic resonance. Finally, in the last part we discuss the use of lipophilic fluorescent probes and imaging techniques as an emerging tool to understand the nano-emulsion droplet stability, surface decoration, release mechanisms, and in vivo fate.
This review is mostly intended for a broad readership and provides key tools regarding the choice of the approach to characterize nano-emulsions. Innovative and uncommon methods will be precious to disclose the information potentially reachable behind a formulation of nano-emulsions, not always known in first intention and with conventional methods.
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