Μm Diameter Beads Research Articles

Experimental and statistical methods for microrheological characterization of heterogeneity in human respiratory mucus mimics of health and disease progression

Human respiratory mucus (HRM) is extremely soft, compelling passive microrheology for linear viscoelastic characterization. We focus this study on the use of passive microrheology to characterize HRM heterogeneity, a phenomenon in normal HRM that becomes extreme during cystic fibrosis (CF) disease. Specifically, a fraction of the mucin polymers comprising HRM phase-separate into insoluble structures, called flakes, dispersed in mucin-depleted solution. We first reconstitute HRM samples to the MUC5B:MUC5AC mucin ratios consistent with normal and CF clinical samples, which we show recapitulate progressive flake formation and heterogeneity. We then employ passive particle tracking with 200 nm and 1 μm diameter beads in each reconstituted sample. To robustly analyze the tracking data, we introduce statistical denoising methods for low signal-to-noise tracking data within flakes, tested and verified using model-generated synthetic data. These statistical methods provide a fractional Brownian motion classifier of all successfully denoised, tracked beads in flakes and the dilute solution. From the ensemble of classifier data, per bead diameter and mucus sample, we then employ clustering methods to learn and infer multiple levels of heterogeneity: (i) tracked bead data within vs. outside flakes and (ii) within-flake data buried within or distinguishable from the experimental noise floor. Simulated data consistent with experimental data (within and outside flakes) are used to explore form(s) of the generalized Stokes–Einstein relation (GSER) that recover the dynamic moduli of homogeneous and heterogeneous truth sets of purely flakelike, dilute solution, and mixture samples. The appropriate form of GSER is applied to experimental data to show (i) flakes are heterogeneous with gel and sol domains; (ii) dilute solutions are heterogeneous with only sol domains; and (iii) flake and dilute solution properties vary with probe diameter.

Real-time impedance-activated dielectrophoretic actuation for reconfigurable manipulation of single flowing particles.

This work presents an innovative all-electrical platform for selective single-particle manipulation. The platform combines microfluidic impedance cytometry for label-free particle characterization and dielectrophoresis for contactless multi-way particle separation. The microfluidic chip has a straightforward coplanar electrode layout and no particle pre-focusing mechanism is required. An original online algorithm analyzes the impedance signals of each incoming particle and regulates in real time the dielectrophoretic voltages according to a desired control logic. As a proof-of-concept, three operation modes are demonstrated on a mixture of 8, 10, and 12 μm diameter beads: (i) particle position swapping across the channel axis, irrespective of particle size, (ii) size-based particle separation, irrespective of particle position, and (iii) sorting of a selected sequence of particles. As a perspective, the versatility of impedance cytometry and dielectrophoresis, and the possibility of configuring alternative control logics, hold promise for advanced particle and cell manipulation.

Continuous Flow Labeling and In-Line Magnetic Separation of Cells

There is an identified need for point-of-care diagnostic systems for detecting and counting specific rare types of circulating cells in blood. By adequately labeling such cells with immunomagnetic beads and quantum dots, they can be efficiently collected magnetically for quantification using fluorescence methods. Automation of this process requires adequate mixing of the labeling materials with blood samples. A static mixing device can be employed to improve cell labeling efficiency and eliminate error-prone laboratory operations. Computational fluid dynamics (CFD) were utilized to simulate the flow of a labeling-materials/blood mixture through a 20-stage in-line static mixer of the interfacial-surface-generator type. Optimal fluid mixing conditions were identified and tested in a magnetic bead/tumor cell model, and it was found that labeled cells could be produced at 1.0 mL/min flow rate and fed directly into an in-line magnetic trap. The trap design consists of a dual flow channel with three bends and a permanent magnet positioned at the outer curve of each bend. The capture of labeled cells in the device was simulated using CFD, finite-element analysis and magnetophoretic mobility distributions of labeled cells. Testing with cultured CRL14777 human melanoma cells labeled with anti-CD146 1.5 μm diameter beads indicated that 90 ± 10% are captured at the first stage, and these cells can be captured when present in whole blood. Both in-line devices were demonstrated to function separately and together as predicted.

Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promote angiogenesis of brain microvascular endothelial cells (BMECs). Angiogenesis is also associated with diseases of the brain due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Here, we develop a high-throughput in vitro blood-brain barrier (BBB) bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier microvessels. A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

Single stream inertial focusing in low aspect-ratio triangular microchannels.

A wide range of microfluidic devices for single stream focusing of cells and particles has emerged in recent years, based on both passive and active methods. Inertial microfluidics offers an attractive alternative to these methods, providing efficient and sheathless passive focusing of cells and beads. Nevertheless, in rectangular microchannels, the presence of multiple equilibrium positions necessitates complicated solutions involving manipulation of the 3D structure in order to achieve single stream flows. Here, we present a new approach to single-stream inertial focusing based on a triangular microchannel geometry. Changing the channel cross-sectional shape leads to asymmetry in the velocity profile, resulting in a size-dependent single stable equilibrium position near the channel apex. We demonstrate that soft lithography masters for such microchannels can be fabricated using PMMA through micromilling, and 15 μm diameter beads can be efficiently focused into a single stream. Confocal microscopy was used to confirm the focusing positions in the microchannel cross-section. We further integrated this device with a laser counting system to form a sheathless flow cytometer and demonstrated the counting of beads with an ∼326 s -1 throughput. The use of a triangular cross-section offers a number of benefits, including simplicity of the fundamental principle and geometry, control of design, a small footprint, and ease of integration, as well as high-precision single position focusing.

Multi-photon vertical cross-sectional imaging with a dynamically-balanced thin-film PZT z-axis microactuator.

Use of a thin-film piezoelectric microactuator for axial scanning during multi-photon vertical cross-sectional imaging is described. The actuator uses thin-film lead-zirconate-titanate (PZT) to generate upward displacement of a central mirror platform, micro-machined from a silicon-on-insulator (SOI) wafer to dimensions compatible with endoscopic imaging instruments. Device modeling in this paper focuses on existence of frequencies near device resonance producing vertical motion with minimal off-axis tilt even in the presence of multiple vibration modes and non-uniformity in fabrication outcomes. Operation near rear resonance permits large stroke lengths at low voltages relative to other vertical microactuators. Highly uniform vertical motion of the mirror platform is a key requirement for vertical cross-sectional imaging in the remote scan architecture being used for multi-photon instrument prototyping. The stage is installed in a benchtop testbed in combination with an electrostatic mirror that performs in-plane scanning. Vertical sectional images are acquired from 15 μm diameter beads and excised mouse colon tissue.

High Spatial Resolution Evaluation of Residual Stresses in Shot Peened Specimens Containing Sharp and Blunt Notches by Micro-hole Drilling, Micro-slot Cutting and Micro-X-ray Diffraction Methods

The moderately high lateral RS gradients (on the order of tens of MPa/μm) near shot peened notches in conjunction with the shallow treatment depth (some hundreds of microns) limit the application of far-field and/or high resolution synchrotron diffraction residual stress measurement techniques. Recently proposed Focused Ion Beam – Scanning Elecron Microscope – Digital Image Correlation (FIB-SEM-DIC) based micro mechanical stress relaxation methods for the measurement of residual stress at the micron scale became suitable techniques for local evaluation of residual stresses and stress gradients in shot peened specimens. In this paper ultra-high resolution (~0.5–0.8 μm depth and 5–10 μm lateral resolution) mechanical relaxation stress measurements were used to evaluate the stress variation local to individual peening dimples in ceramic (60–120 μm diameter beads) shot peened Al-7075-T651 double notched samples having 0.15, 0.5 and 2.0 mm radii using Micro-Hole Drilling (μHD), Micro-Slot Cutting (μSC) and micro X-ray Diffraction (μXRD) methods. The micron-sized sampling volumes enabled the stress to be evaluated in individual impact craters (dimples) showing significant point-to-point variation (~ +/−150 MPa) (with certain dimples even recording tensile stresses). After around 30 μm of layer removal the heavily deformed region had largely been removed and the stress profile became much more homogeneous. At this depth the μHD and μSC results were in good accord with those from μXRD measurements which sample over a much larger volume (~40 μm depth × 50 μm laterally) showing an in-plane compressive stress of around 150 MPa far from the notches with the residual stress rising to about 200 MPa at a blunt (2 mm) notch and 500 MPa for a sharp (0.15 mm) one. Further, these recorded variations of residual stresses were correlated with microstructural features, e.g. grains, networks of sub-surface cracks, intermetallics and highly deformed sub-surface regions, revealed by large volume Serial Sectioning Tomography using Plasma Xe+ Focus Ion Beam – Scanning Electron Microscope (PFIB-SEM), EDS and EBSD maps. This allowed for the first time characterize large volume (100 × 66 × 30 μm3) of shot peened regions with resolution of dozens of nanometers and correlate residual stress depth profiles with 3D microstructural features. Finally, in (Benedetti et al. 2016, Int. J. Fatigue) these RS measurements are used to reconstruct the RS field through finite element (FE) analyses.

Anionic surfaces with minimal charge density can restore burst coagulation of microparticle/exosome-depleted blood plasma

Event Abstract Back to Event Anionic surfaces with minimal charge density can restore burst coagulation of microparticle/exosome-depleted blood plasma Angel Contreras-Garcia1, Desgagné Maxime2, D'Elia L. Noelia3, Charles-Hubert Lafantaisie-Favreau4, Juan Carlos Ruiz5, Georges-Etienne Rivard6, Wertheimer Michel1, Paula Messina3 and Caroline D. Hoemann2, 4 1 Ecole Polytechnique, Engineering Physics, Canada 2 Ecole Polytechnique, Chemical Engineering, Canada 3 Universidad Nacional del Sur, Chemistry, Argentina 4 Ecole Polytechnique, Institute of Biomedical Engineering, Canada 5 Universidad Autónoma Metropolitana-Iztapalapa, Basic Sciences and Engineering, Chemical Engineering, Mexico 6 Hôpital Sainte Justine, Division of Hematology-Oncology, Canada Introduction: Blood contains microparticles (MP) and exosomes derived from a variety of cell types including activated platelets. MPs are procoagulant because their surfaces can express tissue factor and/or calcium-binding sites for coagulation factors that cooperate to induce burst thrombin activation [1]. Platelet-poor plasma (PPP) contains low levels of MPs and can be induced to coagulate after contact with both hydrophilic and hydrophobic surfaces [2]. In a standard thromboelastography (TEG) assay with plastic cups and pins, PPP undergoes spontaneous burst coagulation, as revealed in the TEG trace by a variable clotting time (10 to 26 minutes), a low Angle (~33º) and reproducible low clot tensile strength or Maximum Amplitude (MA ~28 mm) [3] (Fig. 1). PPP coagulates faster and more reproducibly in TEG cups and pins coated with anionic carboxylate-rich nanolayers suggesting FXII activation [2], however the role of MPs is unclear. This study tested the hypothesis that MPs are necessary for PPP burst coagulation in plastic TEG cups, and that anionic surfaces are sufficient to restore burst coagulation of MP-depleted plasma. Materials and Methods: Plastic Cyrolite® TEG cups and pins (Hemoscope, IN, USA) were modified by plasma-enhanced chemical vapor deposition to make hydrophobic (PP-HMDSO, poly(hexamethyldisiloxane) or variable anionic (L-PPE:O, 0, 4.4% or 6.8% carboxylate functional group content) nanocoatings [3]. Coagulation in the TEG cup was initiated by combining i) human citrated PPP (Precision Biologics, NS, Canada), ii) MP-depleted plasma (by ultracentrifugation 150,000xg, 30 min) or iii) PPP 2xenriched for MP, with 20 µL of 200 mM CaCl2 containing 2.5 mg/mL 10 µm diameter beads (latex, borosilicate glass, carboxylated plastic), or 0.25 to 25 mg/mL hydroxyapatite (HA) nanoparticles (2.03 Ca/P ratio) [4]. Bead composition was analyzed by XPS. Statistical tests used ANOVA (Statistica). Results: Glass beads with 9% Si and 4% Ca content triggered the fastest PPP clotting time (p<0.05 vs all hydrophobic surfaces). PPP burst coagulation was enhanced by glass beads, L-PPE:O (6.8% carboxylate), and HA nanoparticles, as shown by the Angle increasing from 36º to >55º. HA nanoparticles accelerated PPP burst coagulation in a dose-dependent manner (p=0.004). MP-depleted plasma failed to clot in plastic TEG cups (Fig. 1), and burst coagulation was progressively restored by L-PPE:O with 4.4 to 6.8% carboxylate content (p<0.001, Fig. 2A). MP-depleted plasma coagulation was also induced by 2.5 mg/mL glass beads and 25 mg/mL HA nanoparticles but not by Latex beads, HMDSO, or 1% carboxylated beads (Fig. 2B). In MP-depleted plasma, L-PPE:O with 6.8% carboxylate content triggered the highest Angle (50º) with a clot tensile strength similar to PPP and MP-enriched PPP (Fig.2B). Discussion: Glass beads, >4.4% carboxylated surfaces and HA were effective MP biomimetics that restored burst coagulation. These results suggest that inorganic anionic surfaces can serve as functional binding sites for the calcium-binding gla domain of factors FIXa, FXa and prothrombin. This study is consistent with the notion that tenase and prothrombinase form side-by-side on the same anionic surface [1]. Conclusions: A minimal 6.8% anionic charge group density is required to bring tenase and prothrombinase in close physical proximity for optimal cooperative behavior. Funding: Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). Salary support was from the Fonds de la Recherche Québec Snaté (FRQ-S), CONACyT (México), MEDITIS Program (NSERC and Ecole Polytechnique), Emerging Leaders in the Americas Program, and The Arthritis Society (Canada)

A size-dependent microparticle capture and release chip using concentric membrane ring barriers

We present a size-dependent microparticle separation chip where different sized slit gaps are formed around a central inlet by membrane ring barriers, concentrically arranged, and adjusted with a single pneumatic source. Previous microparticle separation methods, using multiple filters with different pore sizes, require additional structures and processes to release microparticles after they are captured in the filter. In addition, those methods often result in particle loss due to clogging of the fixed pore filters. We suggest a microparticle separation chip capable of size-dependent capture and release without the particle loss. The present separation chip has four concentrically arranged membrane rings (b1 ∼ b4) with a thickness of 90 μm and widths of 182 μm, 188 μm, 194 μm, and 200 μm, respectively, which form slit gaps estimated to be 11.2 μm, 9.5 μm, 7.6 μm, and 5.8 μm, respectively, at the pneumatic pressure of 80 kPa. In the experiment, we demonstrated microparticle capture and release using two different sizes of PS (polystyrene) beads (diameter = 6.51 ± 0.43 μm and 10.32 ± 0.39 μm) immersed in 0.5% BSA (Bovine Serum Albumin) solution at a flow rate of 100 μl/min. At a pressure of 80 kPa, 10.32 μm and 6.51 μm-diameter beads were captured at ring barriers b3 and b4, respectively. Subsequently, at pressures of 65 kPa and 50 kPa, the 6.51 μm and 10.32 μm-diameter beads were respectively released from the outermost barrier (b4). The capture and release efficiencies of 10.32 μm-diameter beads at the b3 barrier were 91.7 ± 16.7% and 90.9 ± 8.1%, respectively. The purity of 10.32 μm-diameter beads at the b3 barrier was 80.2 ± 6.2%. The capture and release efficiencies of 6.51 μm-diameter beads at the b4 barrier were 100.0 ± 0.0% and 97.1 ± 4.0%, respectively. The purity of 6.51 μm-diameter beads at the b4 barrier was 91.8 ± 2.9%. We have verified that different sizes of captured microparticles were released sequentially by gradually reducing the pressure. The present chip, having concentric membrane ring barriers which can form different sized slit gaps using a single pressure source, is simply capable of not only size-dependent microparticle capture, but also release in size order without particle clogging.

Tunable-angle wedge transducer for improved acoustophoretic control in a microfluidic chip

We present a tunable-angle wedge ultrasound transducer for improved control of microparticle acoustophoresis in a microfluidic chip. The transducer is investigated by analyzing the pattern of aligned particles and induced acoustic energy density while varying the transducer geometry, transducer coupling angle, and transducer actuation method (single-frequency actuation or frequency-modulation actuation). The energy-density analysis is based on measuring the transmitted light intensity through a microfluidic channel filled with a suspension of 5 µm diameter beads and the results with the tunable-angle transducer are compared with the results from actuation by a standard planar transducer in order to decouple the influence from change in coupling angle and change in transducer geometry. We find in this work that the transducer coupling angle is the more important parameter compared to the concomitant change in geometry and that the coupling angle may be used as an additional tuning parameter for improved acoustophoretic control with single-frequency actuation. Further, we find that frequency-modulation actuation is suitable for diminishing such tuning effects and that it is a robust method to produce uniform particle patterns with average acoustic energy densities comparable to those obtained using single-frequency actuation.

A particle trapping chip using the wide and uniform slit formed by a deformable membrane with air bubble plugs

We present a high-efficient particle trapping chip, where a wide and uniform slit is formed by a deformable membrane barrier with air bubble plugs. The previous particle trapping methods based on membrane barriers resulted in low trapping efficiency due to the non-uniform slit gap between the membrane and the substrate, especially at the side walls of rectangular channel. In the present method, the air bubble plugs remained in the extended microchannel during sample filling process, block the particle passage at the both side ends of the membrane, thus all particles flow through the uniform slit gap. Therefore, high-efficient particle trapping without particle loss can be achieved. The present particle trapping chip was composed of three layers: pneumatic (top), membrane and channel (bottom) layers. The membrane was deformed by the pneumatic pressure applied from the top layer. In the experimental study using 10.3 μm-diameter polystyrene beads, the membrane barrier with the air bubble plugs successfully trapped the injected beads with the trapping efficiency of 100% at the flow rate of 10 μl/min, while the barrier without the air bubble plugs showed low efficiency of 20%. At the increased flow rate of 20 μl/min, beads were still trapped with trapping efficiency over 98% in the present device. By using a mixture of 5.7 and 10.3 μm-diameter beads, we also verified the present method was capable to trap and release the beads selectively according to their size with the release efficiency of 95.1%. The present simple and effective particle trapping device is applicable for the high-efficient bioparticle isolation and recovery in the micro total analysis system.

Size based sorting and patterning of microbeads by evaporation driven flow in a 3D micro-traps array

We present a three-dimensional (3D) micro-traps array for size selective sorting and patterning of microbeads via evaporation-driven capillary flow. The interconnected micro-traps array was manufactured by silicon micromachining. Microliters of aqueous solution containing particle mixtures of different sized (0.2 to 20 μm diameter) beads were dispensed onto the micro-traps substrate. The smaller particles spontaneously wicked towards the periphery of the chip, while the larger beads were orderly docked within the micro-traps array.

THE PATH OF CARBON IN PHOTOSYNTHESIS. XXIX. GLASS MICROBEADS

Glass microbeads are introduced as convenient and applicable mechanical supports for hydroponics that can be released from roots to exhibit visually discernible responses. Moreover, glass microbeads refract light, effectively redistributing light toward the phylloplane. The smallest beads, <300 μm diameter, rose from neutral to ∼pH 9 in water and the rise was attributable the raw materials source, sodalime glass. The largest 700 μm diameter beads were relatively stable in buffered nutrients. Photography of unobstructed roots was aided by release of microbeads from ryegrass, coleus, corn, and narcissus. Propagation of ‘Ninsei’ was undertaken in containers constructed with inflow and drainage ports. Photodocumentation of responses to indoxylglucopyranoside exhibited consistent differences as compared to controls when cultured in glass microbeads; and further investigations into increased light intensity by refraction may lead to enhanced photosynthetic efficiency.

Abundance and bacterivory of heterotrophic nanoflagellates in the meromictic Lake Suigetsu, Japan

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 66:149-158 (2012) - DOI: https://doi.org/10.3354/ame01565 Abundance and bacterivory of heterotrophic nanoflagellates in the meromictic Lake Suigetsu, Japan Takahiko Okamura1,*, Yumi Mori1, Shin-ichi Nakano2, Ryuji Kondo3,* 1Graduate School of Bioscience and Biotechnology and 3Department of Marine Bioscience, Fukui Prefectural University, Obama, Fukui 917-0003, Japan 2Center for Ecological Research, Kyoto University, Otsu, Shiga 520-2113, Japan *Emails: ‑s1194001@fpu.ac.jp (T. Okamura), rykondo@fpu.ac.jp (R. Kondo) ABSTRACT: The abundance and bacterivory of heterotrophic nanoflagellates (HNF) were seasonally followed in the oxic and anoxic layers of the meromictic Lake Suigetsu between May 2008 and November 2010. The HNF abundance in the anoxic layer was always lower than in the oxic layer during the study period. Ingestion of fluorescently labeled 0.5 µm diameter beads by the anaerobic HNF in the anoxic layer indicated bacterivory by HNF. The specific ingestion rates in the anoxic layers were generally similar to those taken from the oxic layer, with some exceptions. Our data thus suggested that anaerobic HNF were bacterial consumers with high potential bacterivory comparable to that of aerobic HNF in Lake Suigetsu. Bacterial turnover rates by HNF grazing in the oxic layer were estimated to be as high as ~10% d−1 of the bacterial standing stock. In contrast, the rates in the anoxic layer were <1% d−1 due to the low density of HNF in the anoxic layer. Our data thus provide a valuable contribution to understanding the structure and function of microbial food webs in anoxic aquatic environments. KEY WORDS: Anoxic environments · Grazing rate · Heterotrophic nanoflagellates · Meromictic lake Full text in pdf format PreviousNextCite this article as: Okamura T, Mori Y, Nakano Si, Kondo R (2012) Abundance and bacterivory of heterotrophic nanoflagellates in the meromictic Lake Suigetsu, Japan. Aquat Microb Ecol 66:149-158. https://doi.org/10.3354/ame01565 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 66, No. 2. Online publication date: May 25, 2012 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2012 Inter-Research.

High precision particle mass sensing using microchannel resonators in the second vibration mode

An intrinsic uncertainty in particle mass sensing with the suspended microchannel resonator results from variation in a particle's position near the free end of the resonator. To circumvent this error we employ the second flexural bending mode. This mode exhibits additional frequency peaks while particles pass over the antinode, a point where the frequency shift is insensitive to the lateral position of the particle. We measure polystyrene beads with the first and second modes and confirm that the second mode sensing provides a narrower mass histogram. For 3 μm diameter beads, second mode sensing at the antinode improves the coefficient of variation in buoyant mass from 1.76% to 1.05% for population measurements and from 1.40% to 0.53% for a single trapped particle.

The Copolymer Surfactant P188 Reduces Tension in Permeabilized Cell Membranes

Introduction: Certain biocompatible block copolymer surfactants have been found to have important cytoprotective membrane sealing properties. Resealing of disrupted cell membranes through normal cellular endogenous vesicle- membrane fusion pathways is preceeded by a decrease in membrane line tension. It has been postulated that a similar decrease in membrane line tension preceeds sealing of permeabilized cell membranes by block copolymer surfactants such as Poloxamer 188 (P188). Methods: To test this concept we measured effects of sub-CMC P188 on line-tension in saponin permeabilized plasma membranes of MDCK cells in monolayer cell culture. 10 kDa neutral dextran was used an osmotic control. By recording quasi-static strain laser optical tweezer distraction of membrane-bound antibody coated latex 0.5μm diameter beads, we measured maximum tether length of detergent (0.005% saponin) disrupted MDCK cell membranes before and after treatment with either 0.2mM P188 or 0.2mM 10kDa neutral dextran (ND). Bead selection was determined by position and traction was parallel to cytoskeletal protein orientation. Steady-state tether length for maximum traction force was compared. Results and Discussion: Maximum tether length reflects available membrane reservoir and membrane material properties. Recorded tether lengths were 18.30±2.02 (mean±s.e.m.) μm for normal control cells. 6.46±0.82 μm for cells permeabilized with saponin, 6.18x0.96 μm for permeabilized cells treated with ND, and 11.09±1.09 μm for permeabilized cells treate with P188. Saponin extraction of membrane lipid increases membrane stiffness by reducing reservoir size and creation of water-filled defects.Because it is unlikely that P188 restores membrane reservoir volume, these results suggest P188 reduced membrane tension by resealing water-filled defects.

Synthesis and Single-Molecule Characterization of a DNA Hairpin Construct Based on the TAR RNA Sequence

Optical tweezers are used to examine the folding and unfolding of single molecules, revealing the effects of force, ionic strength, proteins, and small molecules on the kinetic and thermodynamic parameters of bimolecular stability. Here, we examine a DNA hairpin construct related to the trans-activation responsive region (TAR) RNA sequence of HIV-1. In an optical tweezers experiment, a bead at one end of a duplex DNA assembly is attached to a micropipette, while a bead at the other end is held in an optical trap, thus allowing investigators to relate picoNewton forces necessary to unfold the DNA with corresponding nanometer changes in length. Force extension/relaxation data are used to elucidate DNA folding and unfolding processes. Our DNA construct design contains a central hairpin structure with long double-stranded “handles” attached to both the 3’ and 5’ end of the hairpin. The DNA “handles”, each approximately 3 kb in length, were synthesized by PCR using biotin- and digoxigenin-labeled primers that serve to attach the final construct to 5 µm-diameter beads for use with the optical tweezers. The smaller central hairpin sequence was obtained via solid-phase organic synthesis. After purification and assembly with a linker oligonucleotide, the 5’ handle and the hairpin structure were ligated together and purified by agarose gel electrophoresis. This product was then ligated to the 3’ handle and again purified. Individual molecules of the final hairpin construct, approximately 6 kb in length, were bound to streptavidin or anti-digoxigenin-labeled beads and their unfolding behavior was studied using optical tweezers.

Assessment of bovine rod outer segment disk membrane heterogeneity utilizing flow cytometry

The outer segment of rod photoreceptor cells is responsible for initiating visual signal transduction when light levels are low. It consists of stacked disk membranes surrounded by the plasma membrane and is under continuous renewal. Disk membranes are synthesized at the base of the outer segment and are progressively displaced toward the apical tip where they are phagocytosed by the pigment epithelium. This process takes approximately ten days during which time the lipid composition of the disk membrane is modified. Disk membranes become progressively lower in cholesterol and higher in unsaturated phospholipids during spatial displacement. The modification of disk composition is likely important for normal function of the rod cell. Flow cytometry is shown in this study to be a powerful tool to assess differences among rod outer segment disk membranes. First, the feasibility of flow cytometry to detect individual osmotically intact disks was established. Fluorescent beads of 1.0 μm, 0.5 μm, 0.2 μm, and 0.1 μm diameter were used to demonstrate that side-scatter intensity measured on a log scale corresponds to bead diameter. The intensity of disk side scatter predominantly corresponded to 0.1–0.2 μm diameter beads. We next applied this technique to detect caveolin-1, peripherin/rds, and GM1 in disk membranes. Caveolin-1 was detected with FITC-labeled anti-caveolin-1, peripherin/rds with Cy3 labeled anti-peripherin/rds, and GM1 with FITC-labeled cholera toxin subunit B. Fluorescence due to caveolin-1 and peripherin/rds binding was detected in 80% and 70% respectively of the events that corresponded to scattering attributed to disks. Fluorescence attributed to GM1 was detected in 60% of the events that corresponded to disk scattering. The intensity of fluorescence due to caveolin-1 and peripherin/rds labeling was directly proportional to the intensity of side scatter, indicating that both caveolin-1 and peripherin/rds concentrations are uniform among all the disks. Fluorescence intensity of GM1 labeled disks was independent of side-scatter intensity indicating GM1 is heterogeneously distributed among the disks. These results are consistent with integral membrane proteins such as caveolin-1, and peripherin/rds remaining in the disk bilayer as the disks are apically displaced. The GM1 distribution may be similar to the spatial distribution of cholesterol in the outer segment.

Multifunctional protein processing chip with integrated digestion, solid‐phase extraction, separation and electrospray

We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 μm channels for CE, and two serially connected 150 μm deep, 800 μm wide channels packed with 40 to 60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 μg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.

Analysis of systematic error in “bead method” measurements of meteorite bulk volume and density

The Archimedean glass bead method for determining meteorite bulk density has become widely applied. We used well characterized, zero-porosity quartz and topaz samples to determine the systematic error in the glass bead method to support bulk density measurements of meteorites for our ongoing meteorite survey. Systematic error varies according to bead size, container size and settling method, but in all cases is less than 3%, and generally less than 2%. While measurements using larger containers (above 150 cm 3) exhibit no discernible systematic error but much reduced precision, higher precision measurements with smaller containers do exhibit systematic error. For a 77 cm 3 container using 40–80 μm diameter beads, the systematic error is effectively eliminated within measurement uncertainties when a “secured shake” settling method is employed in which the container is held securely to the shake platform during a 5 s period of vigorous shaking. For larger 700–800 μm diameter beads using the same method, bulk volumes are uniformly overestimated by 2%. Other settling methods exhibit sample-volume-dependent biases. For all methods, reliability of measurement is severely reduced for samples below ∼5 cm 3 (10–15 g for typical meteorites), providing a lower-limit selection criterion for measurement of meteoritical samples.