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PubMed2005

Characterization of micromachined ultrasonic transducers using light diffraction tomography.

This paper demonstrates that light diffraction tomography can be used to measure the acoustic field of micromachined ultrasonic transducers (MUT) in cases in which standard methods like hydrophone and microphone measurements fail. Two types of MUTs have been characterized with the method, one air-coupled capacitive MUT (cMUT) and one waterloaded continuous wave (CW) miniature multilayer lead zirconate titanate (PZT) transducer. Light diffraction tomography is an ultrasound measurement method with some special characteristics. Based on the interaction of light and ultrasound, it combines light intensity measurements with tomography algorithms to produce a measurement system. The method offers nonperturbing pressure measurements with high spatial resolution. It has been shown that, under certain circumstances, light diffraction tomography can be used as an absolute pressure measurement method with accuracy in the order of 10% in water and 13% in air. The results show that air-coupled cMUTs in the frequency range of about 1 MHz as well as the extreme near field of a miniaturized CW 10 MHz water-loaded transducer were successfully characterized with light diffraction tomography.

Computer Simulation↗
PubMed2006

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism.

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.

Acyltransferases↗
PubMed2001

Chick cochlear hair cell exocytosis mediated by dihydropyridine-sensitive calcium channels.

1. A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells). 2. Tall hair cell depolarization resulted in changes in cell membrane capacitance (DeltaC(m)) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (I(Ca)) and DeltaC(m) were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around -10 mV. 3. Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed I(Ca) and blocked DeltaC(m). Conversely, the DHP agonist Bay K 8644 increased both I(Ca) and DeltaC(m) amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels.

Animals↗
PubMed2005

A comparison of soil moisture sensors for space flight applications.

Plants will be an important part of future long-term space missions. Automated plant growth systems require accurate and reliable methods of monitoring soil moisture levels. There are a number of different methods to accomplish this task. This study evaluated sensors using the capacitance method (ECH2O), the heat-pulse method (TMAS), and tensiometers, compared to soil water loss measured gravimetrically in a side-by-side test. The experiment monitored evaporative losses from substrate compartments filled with 1- to 2-mm baked calcinated clay media. The ECH2O data correlated well with the gravimetric measurements, but over a limited range of soil moisture. The averaged TMAS sensor data overstated soil moisture content levels. The tensiometer data appeared to track evaporative losses in the 0.5- to 2.5-kPa range of matric potential that corresponds to the water content needed to grow plants. This small range is characteristic of large particle media, and thus high-resolution tensiometers are required to distinguish changing moisture contents in this range.

Biosensing Techniques↗
PubMed2002

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells.

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis.

Animals↗
PubMed2005

Air-coupled through-transmission fan-beam tomography using divergent capacitive ultrasonic transducers.

Abstracttrasonic transducers (CUTs) with curved backplates was used to acquire signals through regions of air containing solid objects, air flow, and temperature fields. Fan-beam datasets were collected and used in a tomographic reconstruction algorithm to produce cross-sectional images of the area under interrogation. In the case of the solid objects, occluded rays from the projections were accounted for using a compensation algorithm and a priori knowledge of the object. A rebinning routine was used to pick out parallel ray sets from the fan-beam data. The effects of further reducing the number of datasets also were investigated, and, in the case of imaging solid objects, characteristic Gibbs phenomena were seen in the reconstructions as expected. However, when imaging temperature and flow fields, the aliasing artefacts were not seen, but the reconstructed values decreased with the size of dataset used. The effect of changing the kernel filter function also was investigated, with the different filters giving the best compromise between image noise, reconstruction accuracy, and amount of data required in each scenario.

Algorithms↗
PubMed2003

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Animals↗
PubMedDate not supplied

Osmotic swelling and hole formation in membranes of thalassemic and spherocytic erythrocytes.

Osmotic swelling and kinetics of the pore formation in the membranes of spherocytic, thalassemic, and normal erythrocytes were studied by measuring the time-dependent capacitance and conductance at a frequency of 0.2 MHz. No significant difference between the swelling rate of control and spherocytic cells was observed, whereas slower kinetics of swelling were found for thalassemic cells. Time records of the conductance indicate that the probability of the pore formation in the stretched membrances varies in the following order: thalassemia < control < spherocytosis. Based on these findings it was concluded that the erythrocyte swelling is controlled by the initial cell shape, volume, intracellular hemoglobin concentration, and elastic membrane properties, whereas the kinetics of the pore formation depend solely on the resistivity of the stretched membrane of the swollen RBC to the osmotic shock. Therefore, it was assumed that investigations of the pore formation may be used not only for examinations of spherocytic and thalassemic cells, but also for normocytic, normochromic, biconcave-shaped RBCs with altered membrane elasticity.

Cell Membrane Permeability↗
PubMed2006

Frequency selectivity of synaptic exocytosis in frog saccular hair cells.

The ability to respond selectively to particular frequency components of sensory inputs is fundamental to signal processing in the ear. The frog (Rana pipiens) sacculus, which is used for social communication and escape behaviors, is an exquisitely sensitive detector of sounds and ground-borne vibrations in the 5- to 200-Hz range, with most afferent axons having best frequencies between 40 and 60 Hz. We monitored the synaptic output of saccular sensory receptors (hair cells) by measuring the increase in membrane capacitance (deltaC(m)) that occurs when synaptic vesicles fuse with the plasmalemma. Strong stepwise depolarization evoked an exocytic burst that lasted 10 ms and corresponded to the predicted capacitance of all docked vesicles at synapses, followed by a 20-ms delay before additional vesicle fusion. Experiments using weak stimuli, within the normal physiological range for these cells, revealed a sensitivity to the temporal pattern of membrane potential changes. Interrupting a weak depolarization with a properly timed hyperpolarization increased deltaC(m). Small sinusoidal voltage oscillations (+/-5 mV centered at -60 mV) evoked a deltaC(m) that corresponded to 95 vesicles per s at each synapse at 50 Hz but only 26 vesicles per s at 5 Hz and 27 vesicles per s at 200 Hz (perforated patch recordings). This frequency selectivity was absent for larger sinusoidal oscillations (+/-10 mV centered at -55 mV) and was largest for hair cells with the smallest sinusoidal-stimuli-evoked Ca2+ currents. We conclude that frog saccular hair cells possess an intrinsic synaptic frequency selectivity that is saturated by strong stimuli.

Animals↗
PubMed2001

Assignment of charge movements to electrogenic reaction steps of Na,K-ATPase by analysis of salt effects on the kinetics of charge movements.

Na,K-ATPase-enriched membrane fragments adsorbed to lipid bilayers were used to study electrogenic Na+ movements induced by enzyme phosphorylation when ATP was photo-released from inactive caged ATP, and simultaneously by externally applied alternating voltages which allowed the measurement of small ATP-induced membrane admittance changes. A detailed analysis of frequency dependence of the capacitance and conductance increments showed that the observed process consists of more than one electrogenic step. The frequency dependence could be described by the sum of two Lorentzian functions and a constant term. The faster process (approximately 2000 s(-1)) was assigned to the release of the first extracellular Na+ ion. The corner frequency of the slower Lorentzian (about 30 s(-1)) coincided with the reciprocal exponential time constant of the falling phase of the transient current, which can be assigned to the conformational transition. Preferentially, the slower process showed a dependence on the ion concentration of choline salts with different anions. The effectiveness of the used chaotropic anions to decelerate the kinetics decreased in agreement with the Hofmeister series, I- > Br- > Cl-. This observation matches their effect on the partition between two phosphoenzyme states of the Na,K-ATPase, as established previously.

Animals↗
PubMed2002

Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

Animals↗
PubMed2005

Capacitive micromachined ultrasonic transducers: fabrication technology.

Capacitive micromachined ultrasonic transducer (cMUT) technology is a prime candidate for next generation imaging systems. Medical and underwater imaging and the nondestructive evaluation (NDE) societies have expressed growing interest in cMUTs over the years. Capacitive micromachined ultrasonic transducer technology is expected to make a strong impact on imaging technologies, especially volumetric imaging, and to appear in commercial products in the near future. This paper focuses on fabrication technologies for cMUTs and reviews and compares variations in the production processes. We have developed two main approaches to the fabrication of cMUTs: the sacrificial release process and the recently introduced wafer-bonding method. This paper gives a thorough review of the sacrificial release processes, and it describes the new wafer-bonding method in detail. Process variations are compared qualitatively and quantitatively whenever possible. Through these comparisons, it was concluded that wafer-bonded cMUT technology was superior in terms of process control, yield, and uniformity. Because the number of steps and consequent process time were reduced (from six-mask process to four-mask process), turn-around time was improved significantly.

Biotechnology↗
PubMed2005

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes.

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca(2+) entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La(3+); (ii) AA (10 microM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and gamma-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca(2+) influx, which is not inhibited by 2-APB or La(3+), indicating that AA activates a second Ca(2+) entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca](i) overload is to cause delayed cell death in astrocytes.

Animals↗
PubMed2002

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse.

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.

Action Potentials↗
PubMed2006

Characterization of mixed alcohol monolayers adsorbed onto a Au(111) electrode using electro-fluorescence microscopy.

A single-crystal Au(111) electrode modified with an adsorbed layer of 1-octadecanol (C18OH) or oleyl alcohol (OLA) in pure or mixed composition was characterized using electrochemical and in situ fluorescence microscopy. Cyclic voltammetry and differential capacitance measurements revealed a repeatable, potential-induced adsorption/desorption process of the surfactant to/from the electrode surface while charge density and film pressure measurements indicated quasi-ideal mixing of the two adsorbed alcohols. A layer less defective than pure C18OH was created with incorporated OLA. Optical characterization was accomplished using epi-fluorescence microscopy combined with electrochemistry (electro-fluorescence microscopy) through the incorporation of two fluorescent probes into the adsorbed surfactant layer. Since molecular luminescence is quenched by a nearby metal, fluorescence was only observed when the fluorescent dye/alcohol layers were desorbed and therefore separated from the metal surface. When desorbed, the structure of the alcohol layers were similar in character, revealing aggregated features which did not change in morphology over numerous desorption/re-adsorption cycles. We have also used the electro-fluorescence technique to estimate the distance separating the metal and desorbed surfactant and believe that the molecules are displaced from the electrode surface by a distance not more than 40 nm.

Adsorption↗
PubMed2005

Design, fabrication and characterization of a capacitive micromachined ultrasonic probe for medical imaging.

In this paper we report the design, fabrication process, and characterization of a 64-elements capacitive micromachined ultrasonic transducer (cMUT), 3 MHz center frequency, 100% fractional bandwidth. Using this transducer, we developed a linear probe for application in medical echographic imaging. The probe was fully characterized and tested with a commercial echographic scanner to obtain first images from phantoms and in vivo human body. The results, which quickly follow similar results obtained by other researchers, clearly show the great potentiality of this new emerging technology. The cMUT probe works better than the standard piezoelectric probe as far as the axial resolution is concerned, but it suffers from low sensitivity. At present this can be a limit, especially for in depth operation. But we are strongly confident that significant improvements can be obtained in the very near future to overcome this limitation, with a better transducer design, the use of an acoustic lens, and using well matched, front-end electronics between the transducer and the echographic system.

Computer-Aided Design↗
PubMed2003

Presynaptic calcium stores modulate afferent release in vestibular hair cells.

Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located approximately 10 microm from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and DeltaCm responses were inhibited after incubation with ryanodine (40 microm) for 8-10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.

Afferent Pathways↗
PubMed2003

Quantitative description of human skin water dynamics by a disposition-decomposition analysis (DDA) of trans-epidermal water loss and epidermal capacitance.

BACKGROUND: In vivo water assessment would greatly benefit from a dynamical approach since the evaluation of common related variables such as trans-epidermal water loss or "capacitance" measurements is always limited to instantaneous data. Mathematical modelling is still an attractive alternative already attempted with bi-exponential empirical models. A classical two-compartment interpretation of such models raises a number of questions about the underlying fundamentals, which can hardly be experimentally confirmed. However, in a system analysis sense, skin water dynamics may be approached as an ensemble of many factors, impossible to discretize, but conceptually grouped in terms of feasible properties of the system. The present paper explores the applicability of this strategy to the in vivo water dynamics assessment. METHODS: From the plastic occlusion stress test (POST) skin water balance is assessed by modelling trans-epidermal water loss (TEWL) and "capacitance" data obtained at skin's surface. With system analysis (disposition-decomposition analysis) the distribution function, H(t), modelled as a sum of exponential terms, covers only the distribution characteristics of water molecules traversing the skin. This may correspond macroscopically to the experimental data accessed by "corneometry". Separately, the hyperbolic elimination function Q(TEWL) helps to characterise the dynamic aspects of water influx through the skin. DISCUSSION AND CONCLUSION: In the observable range there seems to be a linear relationship between the net amount of water lost at the surface by evaporation, and the capability of the system to replenish that loss. This may be a specific characteristic of the system related to what may be described as the skin's "intrinsic hydration capacity" (IHC) a new functional parameter only identified by this strategy. These new quantitative tools are expected to find different applicabilities (from the in vivo skin characterisation to efficacy testing) contributing to disclose the dynamical nature of the skin water balance process.

Adult↗