An asymptotic model of unsteady airway reopening

We consider a simple physical model for the reopening of a collapsed lung airway involving the unsteady propagation of a long bubble of air, driven at a prescribed flow-rate, into a liquid-filled channel formed by two flexible membranes that are held under large longitudinal tension and are confined between two parallel rigid plates. This system is described theoretically using an asymptotic approximation, valid for uniformly small membrane slopes, which reduces to a fourth-order nonlinear evolution equation for the channel width ahead of the bubble tip, from which the time-evolution of the bubble pressure pb* and bubble speed may be determined. The model shows that there can be a substantial delay between the time at which the bubble starts to grow in volume and the time at which its tip starts to move. Under certain conditions, the start of the bubble's motion is accompanied by a transient overshoot in pb*, as seen previously in experiment; the model predicts that the overshoot is greatest in narrow channels when the bubble is driven with a large volume flux. It is also shown how the threshold pressure for steady bubble propagation in wide channels has distinct contributions from the capillary pressure drop across the bubble tip and viscous dissipation in the channel ahead of the bubble.     

A novel concentric double-barrelled calcium-selective microelectrode for small cells

A novel concentric design of double-barrelled Ca2+-selective microelectrode, with an inner pipette tip that protrudes beyond an outer one, has recently been developed and is described. This configuration of pipettes was produced from concentric capillaries in one step using a horizontal pipette puller. For the tip of the inner barrel to protrude, Corning 1724 aluminosilicate glass was selected, as it has a higher melting point than the 1723 glass which is used for the outer barrel. To reduce electrode resistance the inner capillary was best made with a triangular shape. It was preferentially silanized in a dry box by injection of methyltrichlorosilane into only the inner barrel. The Ca2+ neutral carrier-based liquid membrane (ETH 1001) was back-filled from the tip to the shank of the inner pipette and above this CaCl2 solution was added. KCl, which contained EGTA and was buffered to pCa 7, was used to fill the reference barrel. These Ca2+ electrodes showed linear response with slope approximately equal to 30 mV for changes in Ca2+ concentration between 10(-3) and 10(-7) M in the presence of constant [K+]. They offer a number of advantages including a low noise level achieved by the presence of the external concentric KCl electrode, and a simple mechanical structure that allows applications to a variety of small cells.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.     

Quantifying the cleanliness of glass capillaries

I used capillary rise methods to investigate the lumenal surface properties of quartz (fused silica, Amersil T-08), borosilicate (Corning 7800), and high-lead glass (Corning 0010) capillaries commonly used to make patch pipets. I calculated the capillary rise and contact angle for water and methanol from weight measurements. The capillary rise was compared with the theoretical maximum value calculated by assuming each fluid perfectly wetted the lumenal surface of the glass (i.e., zero contact angle, which reflects the absence of surface contamination). For borosilicate, high-lead, and quartz capillaries, the rise for water was substantially less than the theoretical maximum rise. Exposure of the borosilicate, lead, and quartz capillaries to several cleaning methods resulted in substantially better—but not perfect—agreement between the theoretical maximum rise and calculated capillary rise. By contrast, the capillary rise for methanol was almost identical in untreated and cleaned capillaries, but less than its theoretical maximum rise. The residual discrepancy between the observed and theoretical rise for water could not be improved on by trying a variety of cleaning procedures, but some cleaning methods were superior to others. The water solubility of the surface contaminants, deduced from the effectiveness of repeated rinsing, was different for each of the three types of capillaries examined: Corning 7800>quartz>Corning 0010. A surface film was also detected in quartz tubing with an internal filament. I conclude that these borosilicate, quartz, and high-lead glass capillaries have a film on the lumenal surface, which can be removed using appropriate cleaning methods. The surface contaminants may be unique to each type of capillary and may also be hydrophobic. Two simple methods are presented to quantitate the cleanliness of glass capillary tubing commonly used to make pipets for studies of biological membranes. It is not known if the surface film is of importance in electrophysiological studies of biological membranes.     

Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.     

Microvascular and capillary perfusion following glycocalyx degradation.

Systemic parameters and microvascular and capillary hemodynamics were studied in the hamster window chamber model before and after hyaluronan degradation by intravenous injection of Streptomyces hyaluronidase (100 units, 40-50 U/ml plasma). Glycocalyx permeation was estimated using fluorescent markers of different molecular size (40, 70, and 2,000 kDa), and electrical charge. Systemic parameters (blood pressure, heart rate, blood gases) and microhemodynamics (vascular tone, velocity, and blood flow) remained statistically unchanged after injection of hyaluronidase, compared with inactivated hyaluronidase. Conversely, capillary hemodynamics were drastically affected. Functional capillary density, the capillaries perfused with red blood cells (RBCs), decreased by 35%, capillary Hct of the remaining functional capillaries increased from 16 to 27%, and penetration of 70-kDa fluorescent marker increased. Furthermore, plasma-only perfused capillaries statistically increased 30 min after hyaluronidase. The decrease in functional capillary density accounted for an increased RBC flux in the remainder of the capillaries, since the same number of RBCs had to traverse a reduced number of capillaries. Flux balances showed a reduction from baseline of 11% for the RBC flux and 20% for the plasma flux after treatment. These discrepancies are within the margin of error of the techniques used and could be explained by accounting for RBC over-velocity compared with plasma. These findings suggest that the decrease in the glycocalyx leads to capillary perfusion impairments.     

Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

Numerical Simulation of Passage of a Neutrophil through a Rectangular Channel with a Moderate Constriction

The authors have previously presented a mathematical model to predict transit time of a neutrophil through an alveolar capillary segment which was modeled as an axisymmetric arc-shaped constriction settled in a cylindrical straight pipe to investigate the influence of entrance curvature of a capillary on passage of the cell. The axially asymmetric cross section of a capillary also influences the transit time because it requires three-dimensional deformation of a cell when it passes through the capillary and could lead to plasma leakage between the cell surface and the capillary wall. In this study, a rectangular channel was introduced, the side walls of which were moderately constricted, as a representative of axially asymmetric capillaries. Dependence of transit time of a neutrophil passing through the constriction on the constriction geometry, i.e., channel height, throat width and curvature radius of the constriction, was numerically investigated, the transit time being compared with that through the axisymmetric model. It was found that the transit time is dominated by the throat hydraulic diameter and curvature radius of the constriction and that the throat aspect ratio little affects the transit time with a certain limitation, indicating that if an appropriate curvature radius is chosen, such a rectangular channel model can be substituted for an axisymmetric capillary model having the same throat hydraulic diameter in terms of the transit time by choosing an appropriate curvature radius. Thus, microchannels fabricated by the photolithography technique, whose cross section is generally rectangular, are expected to be applicable to in vitro model experiments of neutrophil retention and passage in the alveolar capillaries.     

Functional morphology of capillary bed of the uterus after administration of synestrol

The capillary vessels of rats uterus were examined. The animals who received 0.2% solution were examined 1, 3, 5, 10, 20, 30, 60 days after the last injection. As a marker for capillary vessels the histochemical method for finding the Mg++ adenosine triphosphatase was used. The studies have found prominent qualitative and quantitative changes (activity of enzyme, length and diameter of capillaries) not less than for two months after the last injection of the solution. Particularly prominent changes were found in endometrium between 10-20 days of the restorative period.     

Morphometric study of the blood-carrying capillaries during the administration of a liquid into the salivary gland]

The physiological saline was injected into the excretory duct of the cat parotid gland under the pressure of 30, 70 and 120 cm H2O. It was found with the aid of transmission electronic microscopy, morphometry and statistical analysis that the liquid injection into the gland produces compression of the blood capillaries weaving the acinus. The compression of the capillary tubes is shown by a significant reduction in space given to the lumen of the capillaries and their endothelial layer. The compression of the capillary tubes is combined with a two-fold lessening suggest that the additional blood volume entering the gland in response to its perfusion by the liquid does not reach the blood capillaries and is thrown off into the vein vessels through the shunt communications and that regulation of the blood volume getting into the cat parotid gland capillaries is likely to depend on the hydraulic and osmotic state in the interstitial space of glandular lobes.     

Quantitation of the volume of liquid injected into cells by means of pressure

The amount of TRITC-labeled bovine serum albumin (TRITC-BSA) released from the tip of standardized microcapillaries at different injection pressures and times was investigated. Three test systems were used: (a) formation of droplets of the test solution (TRITC-BSA) in paraffin oil, (b) injection of the test solution into buffer droplets suspended in paraffin oil, and (c) injection into living 3T3 cells. The amount of test substance released was determined by scanning fluorometry. The first procedure (a) allows the fluorometrically determined amount of TRITC-BSA to be related to the volume of released test solution. For this rather large pressures (about 700 hPa) are required to overcome the surface forces counteracting droplet formation. The volume of the spheres was evaluated from photomicrographs of the droplets. The values obtained correlate very well with those determined by measuring the fluorescence emitted by the droplets. Injection into preformed droplets of buffer (b) can be performed with pressures in the range used for injecting cells (50-400 hPa). High reproducibility in the volume released is obtained with a single microcapillary; however, large variations exist between different capillaries, although these should theoretically be of equal diameter. The volume injected into living cells (c) under a given condition may vary by a factor of 5 or more. This variation may be due to viscosity differences of cytoplasm. We recommend, therefore, injection of fluorescent marker substances together with the test substance, enabling the injected volume to be determined by scanning fluorometry or by image analysis.     

Microhemodynamics of blood flow in narrow glass capillaries of 9 to 20 micrometers; the Fahraeus effect

Velocities of the red blood cell (RBC) and the suspending medium in glass capillaries of 9 to 20 micron were measured under microscopic observation. The effects of physical factors such as driving pressure, capillary diameter, hematocrits and RBC deformability on flow velocities were studied using freshly drawn blood of the rat resuspended in phosphate buffered saline solution in the hematocrit range between 5 and 12.5%. These RBC suspensions were made to flow through the test glass capillaries under known negative driving pressures. Ratios of capillary hematocrit to feed hematocrit taken as measures of the Fahraeus effect showed almost constant value of about 0.74. While, ratios of capillary hematocrit to discharge hematocrit showed a characteristic dependence on capillary diameter, showing minimal values at about 13 micron in capillary diameter. The same hematocrit ratios were found to be well correlated with values of wall shear rates estimated from the relative RBC velocities.     

Electrical resistance of a capillary endothelium

The electrical resistance of consecutive segments of capillaries has been determined by a method in which the microvessels were treated as a leaky, infinite cable. A two-dimensional analytical model to describe the potential field in response to intracapillary current injection was formulated. The model allowed determination of the electrical resistance from four sets of data: the capillary radius, the capillary length constant, the length constant in the mesentery perpendicular to the capillary, and the relative potential drop across the capillary wall. Of particular importance were the mesothelial membranes covering the mesenteric capillaries with resistances several times higher than that of the capillary endothelium. 27 frog mesenteric capillaries were characterized. The average resistance of the endothelium was 1.85 omega cm2, which compares well with earlier determinations of the ionic permeability of such capillaries. However, heterogeneity with respect to resistance was observed, that of 10 arterial capillaries being 3.0 omega cm2 as compared with 0.95 omega cm2 for 17 mid- and venous capillaries. The average in situ length constant was 99 micrometers for the arterial capillaries and 57 micrometers for the mid- and venous capillaries. It is likely that the ions that carry the current must move paracellularly, through junctions that are leaky to small solutes.     

Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping

A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.     

Quantifying the geometry of micropipets

Accurate knowledge of the internal diameter (id) of micropipet tips is important, because the ability to study many different aspects of biological membranes is a very sensitive function of tip size. The authors examined two methods used to characterize pipet tips: the digital manometric method (DMM) and bubble number method (BNM). For DMM, the authors compared the ability of Laplace's equation (model I) and a modified form of his equation (model II), which accounts for adhesion between the test fluid and glass Pressure measurements were made with a digital manometer, and ids at the tip were measured using scanning electron microscopy (SEM). The micropipet tips showed a slight asymmetry in id, with a approx 5% difference between maximum and minimum id. On average, model I overestimates the largest id by 2%. Model II overestimates the smaller id by 2%. For micropipet tips ranging from 1.00 to 5.00 μm, the corresponding uncertainties range from 20 to 100 nm. Making the normally hydrophilic glass surface hydrophobic strongly reduced threshold pressures when tested in water, but not 100% methanol. Compared to BNM, DMM was insensitive to changes in atmospheric pressure: BNM can be corrected for changes in atmospheric pressure. Convergence angle(s) can be determined from measurements of the pressure and the axial distance of the meniscus from the tip. The accuracy and precision of digital manometry approaches that of SEM. DMM should be particularly useful in selecting, micropipets for patch clamp studies of small vesicles (<10 μm), and may enable systematic selection of micropipets for many other experiments.     

Some considerations on capillary adjustment to changing metabolism of tissue

Local regulation of blood flow as determined by capillary diameter and the number of open capillaries in a region is considered. The local changes in capillary diameter and in the number of open capillaries are assumed to be due to concentration changes of a diffusible, nonspecified metabolite. This metabolite is produced in the tissue and carried away by the blood stream. Using these assumptions and applying pertinent data on capillaries, deductions are made concerning:

1. (a)
   the law of blood flow as a function of temperature and capillary radius for the hyperemia of high temperature,     
   
   

Follicular microvasculature in the porcine ovary

The microvasculature of porcine ovaries, with special regard to the follicles in the interstitial-stromal tissue, was studied by scanning electron microscopy (SEM) of vascular corrosion casts. Porcine ovaries displayed several coiled arteries in the hilus and many branches with small diameters and a tightly spiraling configuration in the cortical areas. However, small arterioles became straight before entering vascular complexes of follicles and finally divided into capillaries. Vascular baskets of various sizes (150-9,900 micro m in diameter) and architecture related to follicles in various developmental stages were observed in the ovarian cortex. Small follicles (150-300 micro m in diameter) began with a polygonal meshwork of a few large capillary meshes and developed to an obvious spherical microvascular network with a thin single layer of capillaries when reaching 500-700 micro m in diameter. The microvascular architecture of follicles 1,000-2,000 micro m in diameter developed further and had a three-layer vascular plexus. With a diameter of more than 2,000 micro m, the microvasculature of antral follicles was arranged as an inner vascular plexus of about 25 micro m, a middle plexus of about 100 micro m, and an outer capillary plexus of about 30 micro m in thickness. The present observations indicate that follicular vascular baskets of diverse sizes and architecture in various developmental stages support the gradual increase of follicular blood flow during follicle growth in the pig.     

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry

Analyte preconcentration on-line with capillary electrophoresis-3-mass spectrometry (PPC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1–2 mm bed of reversed-phase C₁₈ HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.     

The functional morphology of the uterine capillary bed after synestrol administration

The capillary vessels of rats uterus was examined which were receiving for 7 days 0.2% solution of synoestrol i/m (2.0 mg per 1 kg of body weight). The animals were examined 1, 3, 5, 10, 20, 30, 60 days after the last injection of the solution. As the marker for the capillary vessels the histochemical method for finding the Mg2(+)-adenosintriphosphatase was used. The investigations have found prominent qualitative and quantitative changes (activity of enzyme, length and diameter of capillaries) not less than for two months after the last injection of the solution. Particularly prominent changes were found in endometrium between 10-20 days of the restorative period.