Laser Scanning Confocal Microscopy Experiments Research Articles

Biodegradable core-shell electrospun nanofibers based on PLA and γ-PGA for wound healing

The repair process in wound healing is vital and complicated, which brings great challenge to researchers. Tissue engineering scaffolds provide a realizable approach for wound repair. In this study, nanofibers with Poly(γ-glutamic acid) (γ-PGA) as the core and Polylactic acid (PLA) as the shell materials were prepared via coaxial electrospinning. The effect of flow rate on the structure and diameter of the core-shell nanofibers was investigated. The microstructure and morphology of the core-shell nanofibers were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In laser scanning confocal microscopy experiments (LSCM), core-shell nanofibers loaded with rhodamine and coumarin-6 were excited with red and green light respectively to further investigate the structure of core-shell nanofibers. The hydrophobicity and degradation of core-shell nanofibers were studied by water contact angle test and degradation performance test experiments. The in vitro cell culture study showed favorable biocompatibility of nanofibers. The in vivo animal experiment showed that PLA/γ-PGA core-shell nanofiber membrane was conducive to wound repair, where more than 90% re-epithelialization was observed. It suggested a promising and dependable material for tissue engineering and wound healing.

Influence of Al/Cu content on grain boundary diffusion in Nd-Fe-B magnet via in-situ observation

The influence of aluminum and copper content in the starting Nd-Fe-B magnet on grain boundary diffusion process (GBDP) was studied by observing the phase transformation behaviors of the magnets in-situ at high temperature. A higher coercivity increment is discovered in the sample with higher Al/Cu despite the fact that its Dy diffusion amount is the same as the other. DSC analysis shows an evident melting behavior in the higher Al/Cu sample. Laser scanning confocal microscopy (LSCM) in-situ characterization shows a large amount of melted intergranular phase spills out to the surface simultaneously at around 600 °C in the high Al/Cu sample, while the phase spills out gradually one after another in the range between 623 and 680 °C in the other sample, which indicates that the intergranular phase can be more easily melted in the sample containing more Al/Cu. The area fraction of matrix phase remarkably shrinks while that of intergranular phase enlarges after LSCM heating, which demonstrates the outer region of the Nd2Fe14B grains melt at the temperature of 900 °C. Electron probe microanalyzer result (EPMA) shows that the Nd and Dy concentrate in edge regions and subsequently mix into the intergranular phase with the melting of the grain edge, while a large amount of Al and Cu in the intergranular phase spill out. Nevertheless, the sample with higher starting Al/Cu still remains higher residual contents after LSCM experiments, and that could probably be the main reason why the high Al/Cu magnet shows smaller coercivity decrement after LSCM experiment. Overall, the increase of Al/Cu in the starting magnet optimizes the Dy distribution and the wettability of intergranular phase, enhancing coercivity increment effect further.

Influence of Al2Cu morphology on the incipient melting characteristics in B206 Al alloy

Prevention of incipient melting during solution heat treatment is critical for precipitation hardenable alloys such as B206, since it is deleterious to mechanical properties. For this reason, hot-stage laser scanning confocal microscopy (LSCM) was used for in-situ characterization of incipient melting of blocky Al2Cu and Al-Al2Cu eutectic during transient heating of B206 Al alloy to determine the mechanisms of melting. Microstructural analysis was carried out prior to and following the LSCM experiments using SEM and EDX. The results suggest that the blocky Al2Cu particles gradually developed a rough surface comprised of small spheres of locally melted material in small regions around the particle, followed by complete particle melting. In contrast, the globular Al-Al2Cu eutectic clusters melted completely which was followed by a rapid propagation of liquid film on the sample surface. Following incipient melting, the microstructure consisted mainly of coarse Al-Cu bearing eutectic, small quantities of blocky Al2Cu particles and oxides along grain boundaries.

Role of cholesterol on the transfection barriers of cationic lipid/DNA complexes

Most lipid formulations need cholesterol for efficient transfection, but the precise motivation remains unclear. Here, we have investigated the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphocholine in Chinese hamster ovary cells. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by TE, synchrotron small angle X-ray scattering, and laser scanning confocal microscopy experiments. We prove that cholesterol-containing lipoplexes enter the cells using different endocytosis pathways. Formulations with high cholesterol content efficiently escape from endosomes and exhibit a lamellar-nonlamellar phase transition in mixture with biomembrane mimicking lipid formulations. This might explain both the DNA release ability and the high transfection efficiency. These studies highlight the enrichment in cholesterol as a decisive factor for transfection and will contribute to the rational design of lipid nanocarriers with superior TE.

Time Evolution of Nanoparticle–Protein Corona in Human Plasma: Relevance for Targeted Drug Delivery

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

The Effect of Rho-Associated Kinase Inhibition on the Ocular Penetration of Timolol Maleate

To assess the effects of Rho-associated kinase (ROCK) inhibition on the intraocular penetration of timolol maleate. Ex vivo porcine corneal penetration of timolol maleate, sotalol hydrochloride, or brinzolamide incubated with or without Y-27632 was determined in vertical Franz diffusion cells. The effect of ROCK inhibition on the vasodilation of porcine conjunctival vasculature was assessed by scanning electron microscopy (SEM) and immunohistochemical staining with subsequent laser-scanning confocal microscopy (LSCM). Experiments were conducted in New Zealand White (NZW) rabbits to assess the effect of ROCK inhibition on the intraocular distribution of timolol maleate. ROCK inhibition resulted in minimal alteration of ex vivo porcine corneal drug penetration of timolol, sotalol, or brinzolamide. SEM and LSCM experiments conducted with conjunctiva and sclera tissue in Franz diffusion cells suggested vasodilation in the conjunctival vasculature in the presence of Y-27632. Pretreatment of the eyes of NZW rabbits with Y-27632 resulted in aggregate fold reductions (1 hour, 0.25-fold; 4 hours, 0.45-fold) of timolol maleate drug concentrations in intraocular tissues (aqueous humor, lens, and iris) versus eyes not receiving Y-27632 pretreatment. Pretreatment with a vasoconstrictor, phenylephrine, resulted in a reversal of the effect of Y-27632 on diminished timolol maleate intraocular penetration in NZW rabbits. ROCK inhibition reduced the intraocular penetration of administered timolol maleate presumably due to increased systemic elimination through the conjunctival vasculature. It is anticipated that care in order and timing of ROCK inhibitor administration will be warranted for those patients who may be on a multiple topical drug regimen for primary open-angle glaucoma.

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Ratiometric fluorescent sensors for detecting zinc ions in aqueous solution and living cells with two-photon microscopy

We report two fluorescent Zn 2+ sensors, AD1 and AD2. In aqueous solution, upon addition of Zn 2+, AD1 only showed the enhancement on the fluorescence intensity, while AD2, acting as a ratiometric sensor, demonstrated the variations not only on the fluorescence intensity but also on the emission wavelengths. Both sensors exhibit high sensitivity (nM level) and selectivity for Zn 2+ over Na +, K +, Mg 2+, and Ca 2+ at the millimolar level. Moreover, AD2 undergoes significant increase of the TPA cross sections ( δ max) at 840 nm by the Zn 2+-coordination induced ICT enhancement. The laser scanning confocal microscopy experiments revealed that AD2 is cell-permeable and can indeed visualize the changes of intracellular Zn 2+ in living cells through a ratiometric approach by utilizing two-photon excitation.

Thermal fluctuation forces and wetting layers in colloid-polymer mixtures: Derivation of an interface potential

We discuss wetting layers in phase-separated colloid-polymer mixtures adsorbed at a vertical wall, observed in recent laser scanning confocal microscopy experiments. Matching of colloid and solvent dielectric properties renders van der Waals forces negligible and provides a system governed by short-range forces and thermal fluctuations on which the subtle predictions of renormalization group (RG) theory for wetting can be tested. The width w of the fluid-fluid ("liquid-gas") interface bounding the wetting layer scales with the square root of the wetting layer thickness l, in qualitative agreement with RG theory for short-range complete wetting in three dimensions. The measured wetting layer thickness l as a function of the height h above the horizontal plane of bulk phase separation is compared with two distinct theoretical predictions. A simple heuristic interface potential V(l), first proposed in a previous report, is now fully derived, and confronted here with the interface potential based on the linear RG theory. The heuristic approach does not capture fully the RG treatment. While fundamental differences exist between the two approaches, the resulting predictions for l(h) are almost identical. However, the theory does not follow the precise shape of the experimental curve of l(h).

Hag Mediates Adherence ofMoraxella catarrhalisto Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.