Red Fluorescent Particles Research Articles

VISUALIZATION OF MIXING UPPER AND LOWER STRATIFIED SUSPENSIONS USING RED AND BLUE FLUORESCENT PARTICLES

We conducted visualization experiments to investigate the macroscopic mixing of upper and lower stratified suspensions. We used spectroscopy to distinguish the upper and lower suspensions in the images recorded by a color camera, in which the upper and lower suspended particles were replaced with blue and red fluorescent particles, respectively. We also proposed a method to determine the mass of adsorbed particles and the volume of fluorescent solution required to produce fluorescent particles by using the two calibration curves and the Freundlich equation. Visualization with fluorescent particles proved to be effective for macroscopic mixing by comparing mono and color images. Finally, we found good agreement in the trends between the experimental and numerical results.

Negative pressure ventilation enhances acinar perfusion in isolated rat lungs.

We compared acinar perfusion in isolated rat lungs ventilated using positive or negative pressures. The lungs were ventilated with air at transpulmomary pressures of 15/5 cm H2O, at 25 breaths/min, and perfused with a hetastarch solution at Ppulm art/PLA pressures of 10/0 cm H2O. We evaluated overall perfusability from perfusate flows, and from the venous concentrations of 4-µm diameter fluorescent latex particles infused into the pulmonary circulation during perfusion. We measured perfusion distribution from the trapping patterns of those particles within the lung. We infused approximately 9 million red fluorescent particles into each lung, followed 20 min later by an infusion of an equal number of green particles. In positive pressure lungs, 94.7 ± 2.4% of the infused particles remained trapped within the lungs, compared to 86.8 ± 5.6% in negative pressure lungs (P ≤ 0.05). Perfusate flows averaged 2.5 ± 0.1 mL/min in lungs ventilated with positive pressures, compared to 5.6 ± 01 mL/min in lungs ventilated with negative pressures (P ≤ 0.05). Particle infusions had little effect on perfusate flows. In confocal images of dried sections of each lung, red and green particles were co-localized in clusters in positive pressure lungs, suggesting that acinar vessels that lacked particles were collapsed by these pressures thereby preventing perfusion through them. Particles were more broadly and uniformly distributed in negative pressure lungs, suggesting that perfusion in these lungs was also more uniformly distributed. Our results suggest that the acinar circulation is organized as a web, and further suggest that portions of this web are collapsed by positive pressure ventilation.

Chondrogenic Differentiation of Human Chondrocytes and Stem Cells in Different Cell Culture Systems Using IGF-1-Coupled Particles

Various cell-based therapies use the transplantation of ex vivo cultured chondrocytes or stem cells to support repair of cartilage defects. Cell expansion in vitro is required prior to transplantation accompanied by cell dedifferentiation, resulting in unwanted fibrocartilage formation in vivo. Targeted application of growth factors during in vitro cultivation is intended to enhance chondrogenic differentiation of cells. In previous studies, collagen-based scaffolds enriched with silica particles coupled with the insulin-like growth factor (IGF) 1 were tested, concerning their suitability to increase the in vitro redifferentiation of human chondrocytes. Accordingly, in the present study chondrogenic differentiation potential of IGF-1-coupled particles was investigated using human chondrocytes cultured in scaffold-free spheroid pellet culture. Further, influence of IGF-1-coupled particles on mesenchymal stem cells derived from bone marrow (BM-MSCs) cultured onto collagen–based scaffold or in pellet culture was examined as well pellet culture was examined. Chondrogenic differentiation was induced by the growth factor IGF-1 applied as I) soluble IGF-1 or II) conjugated to red fluorescent silica particles. In addition, control silica particles conjugated with NH2 were used to exclude adverse side effects. Besides cell proliferation, collagen type II and glycosaminoglycan synthesis was quantified and histological staining performed to investigate the chondrogenic differentiation. In pellet culture, IGF-1-coupled particles were applied during the pellet formation only. Traceable red fluorescent particles showed homogenous distribution within the pellets. Adverse effects were not detected. Human chondrocyte pellets displayed significantly increased collagen type II synthesis using IGF-1-coupled particles, compared to soluble IGF-1. Independent of the application mode, induction of chondrogenic differentiation of BM-MSCs cultured in pellets was not suitable with the addition of IGF-1 only. However, BM-MSCs cultivation onto collagen-based scaffold enriched with IGF-1-coupled particle showed superior glycosaminoglycan synthesis, compared to soluble IGF-1 application. Using IGF-1 coupled to particles within a three-dimensional matrix resulted in an increased stimulatory chondrogenic effect, indicating a promising tool for controlled growth factor delivery during treatment of cartilage lesion.

Distribution of particles, small molecules and polymeric formulation excipients in the suprachoroidal space after microneedle injection

The purpose of this work was to determine the effect of injection volume, formulation composition, and time on circumferential spread of particles, small molecules, and polymeric formulation excipients in the suprachoroidal space (SCS) after microneedle injection into New Zealand White rabbit eyes ex vivo and in vivo. Microneedle injections of 25–150 μL Hank's Balanced Salt Solution (HBSS) containing 0.2 μm red-fluorescent particles and a model small molecule (fluorescein) were performed in rabbit eyes ex vivo, and visualized via flat mount. Particles with diameters of 0.02–2 μm were co-injected into SCS in vivo with fluorescein or a polymeric formulation excipient: fluorescein isothiocyanate (FITC)-labeled Discovisc or FITC-labeled carboxymethyl cellulose (CMC). Fluorescent fundus images were acquired over time to determine area of particle, fluorescein, and polymeric formulation excipient spread, as well as their co-localization. We found that fluorescein covered a significantly larger area than co-injected particles when suspended in HBSS, and that this difference was present from 3 min post-injection onwards. We further showed that there was no difference in initial area covered by FITC-Discovisc and particles; the transport time (i.e., the time until the FITC-Discovisc and particle area began dissociating) was 2 d. There was also no difference in initial area covered by FITC-CMC and particles; the transport time in FITC-CMC was 4 d. We also found that particle size (20 nm–2 μm) had no effect on spreading area when delivered in HBSS or Discovisc. We conclude that (i) the area of particle spread in SCS during injection generally increased with increasing injection volume, was unaffected by particle size, and was significantly less than the area of fluorescein spread, (ii) particles suspended in low-viscosity HBSS formulation were entrapped in the SCS after injection, whereas fluorescein was not and (iii) particles co-injected with viscous polymeric formulation excipients co-localized near the site of injection in the SCS, continued to co-localize while spreading over larger areas for 2–4 days, and then no longer co-localized as the polymeric formulation excipients were cleared within 1–3 weeks and the particles remained largely in place. These data suggest that particles encounter greater barriers to flow in SCS compared to molecules and that co-localization of particles and polymeric formulation excipients allows spreading over larger areas of the SCS until the particles and excipients dissociate.

Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Aktphosphorylation. Exposure of hypoxic endothelial cellsto conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.

Droplet-based immunoassay on a 'sticky' nanofibrous surface for multiplexed and dual detection of bacteria using smartphones.

We have developed a rapid, sensitive, and specific droplet-based immunoassay for the detection of Escherichia coli and Salmonella within a single-pipetted sample. Polycaprolactone (PCL) electrospun fibers on indium-tin-oxide (ITO) glass provide a sufficient surface to render a non-slip droplet condition, and while the PCL fibers lend a local hydrophilicity (contact angle θ=74°) for sufficient sub-micron particle adhesion, air pockets within the fibers lend an apparent hydrophobicity. Overall, the contact angle of water on this electrospun surface is 119°, and the air pockets cause the droplet to be completely immobile and resistant to movement, protecting it from external vibration. By using both anti-E. coli conjugated, 510 nm diameter green fluorescent particles (480 nm excitation and 520 nm emission) and anti-Salmonella conjugated, 400 nm diameter red fluorescent particles (640 nm excitation and 690 nm emission), we can detect multiple targets in a single droplet. Using appropriate light sources guided by fiber optics, we determined a detection limit of 10(2) CFU mL(-1). Immunoagglutination can be observed under a fluorescence microscope. Fluorescence detection (at the emission wavelength) of immunoagglutination was maximum at 90° from the incident light, while light scattering (at the excitation wavelength) was still present and behaved similarly, indicating the ability of double detection, greatly improving credibility and reproducibility of the assay. A power function (light intensity) simulation of elastic Mie scatter confirmed that both fluorescence and light scattering were present. Due to the size of the fluorescent particles relative to their incident excitation wavelengths, Mie scatter conditions were observed, and fluorescence signals show a similar trend to light scattering signals. Smartphone detection was included for true portable detection, in which the high contact angle pinning of the droplet makes this format re-usable and re-configurable.

Tomographic PIV measurements of flow patterns in a nasal cavity with geometry acquisition

The flow patterns inside a scaled transparent model of a nasal cavity were measured by tomographic particle image velocimetry (PIV) with three-dimensional (3D) geometry acquisition. The model was constructed using transparent silicone. The refractive index of the working fluid was matched to the index of silicone by mixing water and glycerol. Four cameras and a double-pulse laser system were used for tomographic PIV. Red fluorescent particles and long-pass filters were used to obtain a high signal-to-noise ratio. The complex geometry of the 3D nasal model was acquired by accumulating triangulated 3D particle positions, obtained through a least square-based triangulation method. Certain morphological operations, such as the opening and closing of the nasal cavity, were used to improve the quality of acquired nasal geometry data. The geometry information was used to distinguish the fluid from the solid regions during the tomographic reconstruction procedure. The quality of the model geometry acquisition and tomographic reconstruction algorithms was evaluated using a synthetic image test. Synthetic images were generated by fitting a computational model (stereolithography file) to the virtual 3D coordinates and by randomly seeding particles inside the nasal region. A perspective transformation matrix of each camera was used to generate the synthetic images based on the experimental configuration of the camera. The synthetic image test showed that the voxel reconstruction quality could be improved by applying acquired model geometry in the tomographic reconstruction step. The nasal geometry was acquired and a flow velocity field was determined by cross-correlating the reconstructed 3D voxel intensities.

Reciprocating flow-based centrifugal microfluidics mixer

Proper mixing of reagents is of paramount importance for an efficient chemical reaction. While on a large scale there are many good solutions for quantitative mixing of reagents, as of today, efficient and inexpensive fluid mixing in the nanoliter and microliter volume range is still a challenge. Complete, i.e., quantitative mixing is of special importance in any small-scale analytical application because the scarcity of analytes and the low volume of the reagents demand efficient utilization of all available reaction components. In this paper we demonstrate the design and fabrication of a novel centrifugal force-based unit for fast mixing of fluids in the nanoliter to microliter volume range. The device consists of a number of chambers (including two loading chambers, one pressure chamber, and one mixing chamber) that are connected through a network of microchannels, and is made by bonding a slab of polydimethylsiloxane (PDMS) to a glass slide. The PDMS slab was cast using a SU-8 master mold fabricated by a two-level photolithography process. This microfluidic mixer exploits centrifugal force and pneumatic pressure to reciprocate the flow of fluid samples in order to minimize the amount of sample and the time of mixing. The process of mixing was monitored by utilizing the planar laser induced fluorescence (PLIF) technique. A time series of high resolution images of the mixing chamber were analyzed for the spatial distribution of light intensities as the two fluids (suspension of red fluorescent particles and water) mixed. Histograms of the fluorescent emissions within the mixing chamber during different stages of the mixing process were created to quantify the level of mixing of the mixing fluids. The results suggest that quantitative mixing was achieved in less than 3 min. This device can be employed as a stand alone mixing unit or may be integrated into a disk-based microfluidic system where, in addition to mixing, several other sample preparation steps may be included.

A microfluidic chaotic mixer using ferrofluid

A novel micromixer which utilizes chaotic mixing induced by ferrofluid actuation is developed. The micromixer is fabricated by a polydimethylsiloxane micromolding technique. The micromixer consists of a T-shaped main mixing channel and two parallel sub-channels that intersect the main channel. Oscillation of a couple of ferrofluid slugs in the sub-channels, induced by external permanent magnet actuation, generates chaotic advection in the main channel flow. To visualize the mixing, red fluorescent particles are supplied to one of the inlets of a T-shaped channel, and mixing flow is observed by a fluorescence microscope and an intensified CCD camera. The mixing experiments are performed with various ferrofluid perturbation frequencies and main stream flow velocities. The optimal mixing conditions determining the upper and lower limits of the most effective Strouhal numbers are found from the experimental results.

Further studies on the nature of red fluorescent structures in neuroblastoma monolayer cells vitally stained with acridine orange.

The nature of red fluorescent particles in vitally acridine orange stained C 1300 neuroblastoma monolayer cells was evaluated by electron microscopy, cytofluorometry, cytopharmacological and cell fractionation studies. At the ultrastructural level the distribution of red fluorescent granules correlated with that of the Golgi complex and Golgi derived structures during various stages of differentiation, mitosis, and under colcemid treatment. Cytopharmacological studies revealed that red fluorescence was displaced in a concentration and time dependent manner with the basic drugs chloroquine and quinacrine. Subcellular fractionation studies showed that acridine orange was concentrated in fractions that also contained the highest amount of acid phosphatase and electron dense vesicles. Vital acridine orange staining of neuroblastoma cells in culture can give information on the relationship between Golgi-derived vesicles and cell functions like proliferation and differentiation. The influence of drugs on these processes can be studied.