Fluorescence imaging of electrical activity in cardiac cells using an all-solid-state system

Tracking spatial and temporal determinants of cardiac arrhythmogenesis at the cellular level presents challenges to the optical mapping techniques employed. In this paper, we describe a compact system combining two nontraditional low-cost solutions for excitation light sources and emission filters in fluorescence measurements of transmembrane potentials, Vm, or intracellular calcium, [Ca2+]i in cardiac cell networks. This is the first reported use of high-power blue and green light emitting diodes (LEDs), to excite cell monolayers stained with Vm - (di-8-ANEPPS) or [Ca2+]i - (Fluo-3) sensitive dyes. In addition, we use simple techniques for fabrication of suitable thin emission filters with uniform properties, no auto-fluorescence, high durability and good flexibility for imaging Vm or [Ca2+]i. The battery-operated LEDs and the fabricated emission filters, integrated with a fiber-optic system for contact fluorescence imaging, were used as tools to characterize conduction velocity restitution at the macro-scale. The versatility of the LEDs for illumination is further emphasized through 1) demonstration of their usage for epi-illumination recordings at the single-cell level, and 2) demonstration of their unique high-frequency light modulation ability. The LEDs showed excellent stability as excitation light sources for fluorescence measurements; acceptable signal-to-noise ratio and negligible cell photodamage and indicator dye photobleaching were observed.     

A dynamic action potential model analysis of shock-inducedaftereffects in ventricular muscle by reversible breakdown of cellmembrane

To elucidate the subcellular mechanism underlying the aftereffects of high-intensity dc shocks, a small pore, which mimics reversible breakdown of the cell membrane (electroporation), was incorporated into the phase-2 Luo-Rudy (L-R) model of ventricular action potentials. The pore size was set to occupy 0.15%-4.25% of the total cell membrane during the 10-ms shock. The pore was assumed to decrease after the shock exponentially with a time constant of 100-1,400 ms to simulate resealing process. In normal myocytes, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by prolonged depolarization and oscillation of membrane potential like early afterdepolarization (EAD). Time- and voltage-dependent changes in the delayed rectifier K+ currents (IKr, IKs) in combination with those of L-type Ca2+ current (ICa,(L)) and ion flux through the pore (I(pore)) are responsible for the potential changes. Spontaneous excitation from the oscillation depends on activation of ICa,(L). In myocytes overloaded with Na+ and Ca2+ secondary to 90% inhibition of Na+-K+ pump, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by slower cyclic depolarization in response to spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). This delayed afterdepolarization-type oscillation is abolished by complete block of Ca2+ release from the SR. These findings suggest that high-intensity electric field application will cause arrhythmogenic responses through a transient rupture of sarcolemma with different subcellular events in ventricular cells under normal and pathological conditions.     

利用膜片钳-激光扫描共聚焦显微镜同步实时系统观察心肌细胞浆内钙离子的释放

目的:采用膜片钳和激光扫描共聚焦显微镜同步实时系统观察心肌细胞钙离子的释放.方法:在体外培养的单个心室肌细胞上进行全细胞模式膜片钳技术和共聚焦显微镜钙成像技术相结合,同步实时记录钙火花和钙离子浓度变化.结果:在全细胞模式膜片钳记录心肌细胞膜钙电流的同时,激光扫描共聚焦显微镜可准确记录到胞浆内出现的钙火花.此技术有可能对于明确钙火花的特征.准确理解兴奋一收缩偶联的微观机制有重要意义.     

Mechanism of Abnormal Sarcoplasmic Reticulum Calcium Release in Canine Left-Ventricular Myocytes Results in Cellular Alternans

Electrocardiographic alternans are known to predispose to increased susceptibility to life threatening arrhythmias and sudden cardiac death. While deficiencies in Ca²⁺ transport processes have been implicated in the genesis of cellular alternans, the underlying mechanisms have been elusive, and are the goal of this study. A novel reverse engineering approach that applies a simultaneous action potential (AP) and [Ca²⁺ ]i clamp of experimentally obtained data, to a previously described left-ventricular canine myocyte model, is employed to isolate the molecular and cellular mechanisms underlying cardiac alternans. The model-derived sarcoplasmic reticulum (SR) Ca²⁺ in control beats (102.1 plusmn 12.9 nM, n = 639 ), although larger, is not statistically significantly different as compared to beats corresponding to small [Ca²⁺ ]_i (99.3 plusmn 35.4 nM, n = 310, p = NS), but is significantly smaller as compared to beats corresponding to large [Ca²⁺ ]_i (122.6 plusmn 31.0 nM, n = 311, p<0.000001) during alternans. The model indicates that the increased SR Ca²⁺ in these beats triggers multiple ryanodine receptor (RyR) channel openings and delayed Ca²⁺ release that subsequently triggers an inward depolarizing current, a subthreshold early after depolarization, and AP prolongation. In conclusion, the results presented in this study support the idea that aberrant RyR openings on alternate beats are responsible for the [Ca²⁺ ]_i alternans-type oscillations, which, in turn, give rise to AP alternans.     

Voltage-dependent effects of Ca(2+)/calmodulin on Cl(-) channel in cardiac sarcoplasmic reticulum

A new kind of chloride channels in the cardiac sarcoplasmic reticulum, 116 pS Cl(-) channel (500 mM Cl(-) in the cis and 50 mM Cl(-) in the trans chamber solutions), which is activated by protein-kinase-A-dependent phosphorylation, has been determined to conduct adenine nucleotide as a transporter between cytosol and SR lumen. We investigated the voltage-dependent gating of this Cl(-) channel by recording single-channel activities using the planar lipid bilayer-vesicle fusion technique. The channel activities did not change at different membrane potentials (-100 mV to +50 mV) or different Ca(2+) concentrations (1 nM to 1 mM) in cis solution. In the presence of calmodulin (CaM) (0.1 microM /microg SR vesicles), however, Ca(2+) added to the cis solution at 0 mV inhibited channel openings in a Ca(2+) -concentration-dependent manner. These effects were prevented by the addition of CaM inhibitors. The blocking effects of CaM differed depending on the membrane potentials at negative potentials below -20 mV. With CaM and 3 microM Ca(2+), the values of opening probability were 0 at -80 mV, 0.2 at -40 mV, 0.3 at -20 mV, 0.71 at 0 mV and 0.92 at +20 mV. These results may indicate the membrane potential affects the action of Ca(2+) /CaM complex     

Application of real-time confocal laser scanning microscopy to observe living cells in tissue specimens

Fluorescence microscopy imaging has developed into an important tool for the study of cell structure and function in cell biology. This non-invasive technique permits the characterization, localization and qualitative quantification of free ions, messengers, pH, voltage and other molecules in living cells. The regulation of cytosolic Ca2+ homeostasis is essential for cells. However, most investigations have used cultured or isolated cells as an experimental model and, consequently, provide only limited insight into the mechanisms that operate in tissue in situ. More useful information could be obtained by studying intact tissue specimens. The calcium dynamics of some tissue specimens, such as arteriole smooth muscle cells, supra cervical ganglia and peripheral nerve bundles, were analysed in this study. Real-time confocal microscopy revealed that individual cells exhibited different [Ca2+]i dynamics and the responses to transmitters/modulators were heterogeneous. It is important that the confocal microscopes have good detection performances, due to the reduction of stray light. We conclude that real-time confocal microscopy is a useful tool for investigating structural and functional changes of cells in living tissues, although suitable tissue-preparation is important for these measurements.     

Multiscale modeling of calcium dynamics in ventricular myocytes with realistic transverse tubules

Spatial-temporal Ca(2+) dynamics due to Ca(2+) release, buffering, and reuptaking plays a central role in studying excitation-contraction (E-C) coupling in both normal and diseased cardiac myocytes. In this paper, we employ two numerical methods, namely, the meshless method and the finite element method, to model such Ca(2+) behaviors by solving a nonlinear system of reaction-diffusion partial differential equations at two scales. In particular, a subcellular model containing several realistic transverse tubules (or t-tubules) is investigated and assumed to reside at different locations relative to the cell membrane. To this end, the Ca(2+) concentration calculated from the whole-cell modeling is adopted as part of the boundary constraint in the subcellular model. The preliminary simulations show that Ca(2+) concentration changes in ventricular myocytes are mainly influenced by calcium release from t-tubules.     

Impact of on- and off-chip protection on the transient-induced latch-up sensitivity of CMOS IC

Measurements and mixed-mode simulations are used for the analysis of transient-induced latch-up (TLU) in CMOS IC. The transient interaction of the parasitic SCR with the surrounding off-chip and on-chip circuitry is investigated during positive and negative system-level ESD stress. It is shown, that sufficient on-chip decoupling and an active clamp can improve the TLU robustness of a circuit.     

小鼠精子获能及顶体反应过程中Ca^2＋及Ca^2＋—ATPase的研究

利用焦锑酸钾电镜定位Ｃａ＾２＋的技术，及铅捕获法定位细胞Ｃａ＾２＋－ＡＴＰａｓｅ的技术，研究小鼠了获能及顶体反应过程中Ｃａ＾２＋及Ｃａ＾２＋－ＡＴＰａｓｅ的变化，结果表明，获能前精子顶体囊外缘有Ｃａ＾２＋分布，顶体膨胀时，Ｃａ＾２＋消失，开始顶体反应时，顶体外膜，顶体囊泡及顶体后区有Ｃａ＾２＋分布，顶体反应后，仅顶体后区有Ｃａ＾２＋分布精子尾部线粒体及轴丝始终有Ｃａ＾２＋分布。     

2.4GHz低噪声放大器的全芯片ESD保护设计

设计并流片验证了一种0.18μmRFCMOS工艺的2.4GHz低噪声放大器的全芯片静电放电(ESD)保护方案。对于射频(RF)I/O口的ESD防护,主要对比了二极管、可控硅(SCR)以及不同版图的互补型SCR,经流片与测试,发现岛屿状互补型SCR对I/O端口具有很好的ESD防护综合性能。对于电源口的ESD防护,主要研究了不同触发方式的ESD保护结构,结果表明,RCMOS触发SCR结构(RCMOS-SCR)具有良好的ESD鲁棒性和开启速度。基于上述结构的全芯片ESD保护设计,RF I/O口采用岛屿状布局的互补SCR结构的ESD防护设计,该ESD防护电路引入0.16dB的噪声系数和176fF的寄生电容,在人体模型(HBM)下防护能力可达6kV;电源口采用了RCMOS-SCR,实现了5kV HBM的ESD保护能力,该设计方案已经在有关企业得到应用。     

低功率激光对细胞质膜通透性及细胞功能的影响

目的:探索低功率激光对细胞质膜通透性及细胞功能的影响。方法:以波长为632.8nm,功率密度为5.4mW/cm~2的氦氖激光照射人外周血淋巴细胞15、30、60分钟,并采用钙荧光指示剂Fura—2/Am定量测试法检测淋巴细胞内游离钙浓度和质膜Ca~(2+)—Mg~(2+)—ATP酶活性变化。结果:照射后淋巴细胞内游离钙浓度明显低于正常(P<0.05);同时细胞膜Ca~(2+)—Mg~(2+)—ATP酶活性增加(P<0.05);而且照射后细胞内游离钙浓度降低与质膜上Ca~(2+)—Mg~(2+)—ATP酶的激活呈负相关。结论:低功率激光照射激活细胞膜Ca~(2+)—Mg~(2+)—ATP酶活性,使细胞膜对钙通透性发生变化,且影响到细胞内Ca~(2+)贮存,造成细胞膜通透性和细胞功能的改变。     

Dark Currents in a Fully-Depleted LWIR HgCdTe <Emphasis Type="Italic">P</Emphasis>-on-<Emphasis Type="Italic">n</Emphasis> Heterojunction: Analytical and Numerical Simulations

In this paper, we use the theory of Evans and Landsberg, which is a generalization of the Shockley–Read–Hall recombination statistics in the space charge region (SCR), to include effects of Auger and radiative recombination processes that are also of origin in the SCR. Using analytical expressions for the current density, we calculate the total dark current density for a variety of conditions. Contributions include radiative and Auger transitions of origin in both the quasi-neutral region and the SCR. Numerical simulations are used to assess the nature of the limitations associated with the analytical calculation in the n-extrinsic region (\(N_{\rm d} \gg n_{\rm i}\), where \(N_{\rm d}\) is the doping concentration and \(n_{\rm i}\) is the intrinsic carrier concentration), and to extend the calculations to operating temperatures in the intrinsic region (\(n_{\rm i} \gg N_{\rm d}\)). Major findings include the observation that in a fully depleted \(P^+n\) double-layer planar hetero-structure, at a reverse bias voltage sufficiently high to suppress the Auger process, SRH centers are not limiting, and the dark current is due to radiative transitions of origin in the n-side SCR. From the numerical simulations, while the Auger recombination rate changes drastically with varying the carrier concentration (such as applying reverse bias), the radiative recombination rate remains nearly invariant to varying the carrier concentration, and, as such, does not appreciably change with increasing reverse bias. Using the theory of van Roosbroeck and Shockley, the radiative recombination rate is obtained by integrating the measured optical absorption coefficient over all photon energies. Hence, the theory links the measured absorption coefficient to the measured dark current density for conditions in which the dominant current component is due to radiative recombination. Finally, the numerical simulations reveal, in both the n-extrinsic and intrinsic operating regions, that, under sufficient conditions, the detector is radiatively limited.     

ESD protection design for I/O cells with embedded SCR structure as power-rail ESD clamp device in nanoscale CMOS technology

This paper presents a new electrostatic discharge (ESD) protection design for input/output (I/O) cells with embedded silicon-controlled rectifier (SCR) structure as power-rail ESD clamp device in a 130-nm CMOS process. Two new embedded SCR structures without latchup danger are proposed to be placed between the input (or output) pMOS and nMOS devices of the I/O cells. Furthermore, the turn-on efficiency of embedded SCR can be significantly increased by substrate-triggered technique. Experimental results have verified that the human-body-model (HBM) ESD level of this new proposed I/O cells can be greater than 5 kV in a 130-nm fully salicided CMOS process. By including the efficient power-rail ESD clamp device into each I/O cell, whole-chip ESD protection scheme can be successfully achieved within a small silicon area of the I/O cell.     

Modeling the IEC 61000-4-4 EFT Injection Clamp

The paper analyzes the test setup required by the International Electrotechnical Commission (IEC) 61000-4-4 to evaluate the immunity of electronic equipment to electrical fast transients (EFTs), and proposes an electrical model of the capacitive coupling clamp, which is employed to add disturbances to nominal signals. The study points out limits on accuracy of this model, and shows how it can be fruitfully employed to predict the interference waveform affecting nominal system signals through computer simulations.      

Extracellular Ca2+ sensitivity of mGluR1alpha associated with persistent glutamate response in transfected CHO cells

We previously reported that the metabotropic glutamate receptor 1alpha (mGluR1alpha) can be activated not only by glutamate but also by extracellular Ca2+ (Ca2+o), and that Ser 166 in the extracellular domain determines the sensitivity to Ca2+o. In the present study, we investigated by intracellular Ca2+ (Ca2+i) imaging, the effect of Ca2+o on the glutamate responses of Chinese Hamster Ovary (CHO) cells stably expressing mGluR1alpha wild-type (CHO-wt). As a negative control, we carried out similar experiments using CHO cells expressing Ser166Asp mutant of mGluR1alpha (CHO-S166D) or the substance P receptor (CHO-SPR), which were not activated by Ca2+o application. We observed a remarkable prolongation of the duration of the glutamate response in CHO-wt cells in a Ca2+o concentration dependent manner. In CHO-S166D cells and CHO-SPR cells, only a small sustained component of the glutamate response was observed in the presence of Ca2+o. These sustained components were blocked by SKF-96365, a blocker of receptor-operated Ca2+-influx. Thus, it was concluded that the Ca2+o-sensing function of mGluR1alpha-wt induced the persistent opening of the receptor-operated Ca2+-permeable channels, probably by persistent activation of the receptor by glutamate. We additionally observed that the dose-response relationship of CHO-S166D and CHO-SPR shifted significantly by changing Ca2+o concentration, i.e. Ca2+o was required to maintain the normal ligand responses of these receptors.     

Evidence for roles of the activating function in electric stimulation

The hypothesis that the activating function drives transmembrane voltage changes (delta Vm) has been tested in hearts. Optical delta Vm were measured during activating functions produced with nonuniform and uniform transparent electrodes. When a nonuniform electrode was used to produce [equation: see text], the signs of delta Vm and [equation: see text] matched. The extracellular voltage gradients, often assumed important, did not predict delta Vm. When a uniform electrode was used to eliminate [equation: see text], the signs of delta Vm matched the signs of [equation: see text] estimated from variations in heart width. Demonstration of the activating function as a determinant of stimulation may improve research and therapy that use electric stimulation.     

Fast XYT imaging of elementary calcium release events in muscle with multifocal multiphoton microscopy and wavelet denoising and detection

We used multifocal multiphoton microscopy to image fast, localized elevations of the cytosolic Ca2+ concentration in two spatial dimensions plus time (XYT). This technique extends the common spatially 1-D XT imaging and allows the acquisition of more than ten times longer time series (>500 images) and ten times larger areas of interest than for previously used confocal XYT imaging techniques due to lower phototoxicity and fast multifocal scanning. We recorded spontaneously occurring elementary Ca2+ release events in chemically permeabilized adult mammalian skeletal muscle fibers using two-photon excitation of the fluorescent dye Fluo-4. The resulting time series were analyzed with an automated denoising and detection algorithm based on the à trous implementation of the discrete wavelet transform. Wavelet coefficient hard-thresholding is used for denoising and event detection is performed across several wavelet scales. The spatiotemporal characteristics of the detected Ca2+ release events are followed throughout the XYT stack and are parametrized using a biophysically valid anisotropic Gaussian event model. The proposed method allows a detailed spatiotemporal analysis of elementary Ca2+ release events underlying the excitation-contraction coupling process in muscle.     

兔骨骼肌ryanodine受体/钙释放通道的AFM研究

Ryanodine受体（RyR)是位于细胞内质网膜上的钙释放通道,在肌细胞的兴奋－收缩偶联中发挥重要作用。RyR难于结晶,以往主要是用电镜和计算机三维重组相结合的方法来获得其高级结构的信息。由于RyR结构庞大,易于水解,在提取的过程中需要加入外源性磷脂以保护其结构,而磷脂的存在对电镜的分辨率有影响,所以电镜研究的RyR在提 过程中一般不添加外源性磷脂,这样得到的结构信息并没有反映RyR在生理状况下的真实情况,我们用原子力显微镜（AFM）对RyR进行研究,经过了空气中RyR成像,液体中RyR成像,重组到脂双层中的RyR研究几个阶段,提供了RyR在接近生理条件下的结构信息,并为进一步研究其结构和功能的关系打下了基础。     

Presenilin1基因对心肌细胞肌浆网钙容量的影响

目的:探讨Presenilin1基因对心肌细胞肌浆网钙容量的影响.方法:本实验以AAV-9为载体构建心肌特异性PS1-shRNA重组腺相关病毒,通过尾静脉注射PS1-shRNA重组腺相关病毒干扰大鼠心肌组织中Presenilin1基因的表达,并利用激光扫描共聚焦显微镜检测并记录心肌细胞肌浆网钙容量及舒张期自发钙释放事件的变化;同时,采用western blot技术分析各组大鼠心肌组织中雷诺丁受体及钙泵表达水平的改变.结果:与对照组相比,PS1-shRNA重组腺相关病毒干预组大鼠心肌细胞肌浆网钙容量显著降低,自发钙释放事件明显增加;雷诺丁受体表达水平略降低,钙泵表达水平无显著性变化.结论:Presenilin1基因表达水平降低会引起心肌细胞钙泄漏,从而造成心肌细胞肌浆网钙容量降低.     

Translation invariance and sampling theorem of wavelet

The sampling theorem for wavelet spaces built by Walter (1992) lacks the translation invariance except for Walter's weak translation invariant wavelet, i.e., Meyer's wavelet. Indeed, we must know a priori the shift offset a in the samples {f(n+a);n∈Z}; otherwise, the waveform cannot be recovered since the interpolation function is dependent on this offset. In this correspondence, we generalize our metric functional to metrize weak shiftability and find a somewhat surprising result that the B spline wavelets of order n⩾3 are degenerate shiftable. Thus, we can recover approximately the waveform by double sampling without any information on shift offset a