细胞内微电极尖的削磨

细胞内微电极的应用日益增多,如研究神经细胞膜的电学性质及其离子通透情况,尤其是近年来建立离体脑片技术得以进行较长时间稳定的细胞内放电记录,更使学者乐于应用这种电极进行探索。细胞内微电极要求其尖端很细而且锐利,有利于刺入细胞并使细胞损伤减至最低程度。但微电极的电阻主要在其尖端,尖端变细则电阻急剧上升。电阻增大又使噪音变大,并因负电容补偿困难而导致波形失真。电阻超过80mΩ的微电极其电学性质趋于不稳,通过这种微电极向细胞内注入电流也成问题。目前较好的解决办法是将制好的微电极尖端加以斜磨,使其尖端既尖锐而电阻又小,便于穿刺细胞膜,且不易阻塞。由于斜磨口不大,也不存在     

原代单层培养的大鼠搏动心肌细胞内电位的记录方法

自发性节律搏动是原代单层培养心肌细胞的一个重要特征。培养心肌细胞的平均直径较小,自发性节律搏动频率较快。用玻璃微电极记录培养心肌细胞的电位,穿刺成功率较低,且难以稳定在细胞内。我们采用加硅涂层的玻璃微电极方法能在单层搏动培养心肌细胞内稳定地记录1h以上。 所用玻璃微电极是由内含玻璃纤维,外径为1mm的毛细管加热后,经两次拉曳,得到颈长15mm,尖端直径<0.1μm,长度为2～3μm的微电极。并在管内灌入3mol/L KCl溶液,直流电阻为30～50MΩ。在微电极灌注电解质液后,将其尖端浸在含2％二甲基二氯硅烷的四氯化碳溶液内硅化数秒钟。     

一种新型金属微电极的制备方法

在利用微电极技术进行动物脑功能研究的急性和慢性实验中,常用环氧树酯绝缘漆作钨丝微电极的绝缘。此方法程序繁琐,电极绝缘性能较差,重复使用率低。我们结合自己的经验进行了绝缘的金属微电极探索,现扼要介绍在钨丝尖端紧包一薄层玻璃的微电极制备步骤。 1.准备直径为200～250μm、长10cm的挺直钨丝及长7cm、外径2mm、内径0.6mm的GG17厚壁玻璃毛细管。 2.把多根钨丝的一端排齐夹好,成排地浸入75％     

制备玻璃绝缘钨丝微电极时的阻抗控制和记录神经元发放时对脑波动的控制

1.本文介绍一种改进的毛细玻管-钨丝微电极的制备法,主要之点是将事先制备好的钨丝尖端全部密封于玻管内,然后用氟氢酸溶去尖端包被之玻璃以控制钨丝尖端裸露之程度。与钨丝尖端之粗细选择相配合则可能获得各种不同之阻抗,其范围为1-10MΩ。记录细胞外发放的适宜阻抗为2-4MΩ。2.应用此种电极,既可记录小脑皮层较大的浦肯野氏细胞又可记录较小的颗粒细胞发放而不拾取穿行于其间的轴突或树突的活动。此种电极具有良好的稳定性和较低的噪音水平并具较大的穿刺强度和较少损伤脑组织。制备方便,不易损坏,可多次反复使用。3.利用在微电极推进仪上增设的金属环,有效地控制了脑波动,从而有可能更准确地记录各深度的细胞活动。金属环经绝缘连接后,可作为记录刺入部周围脑皮层诱发电位之电极。4.利用钨丝电极,选适宜之泸波参数与阻抗尚可记录该刺入深度之诱发电位。     

磨针仪的自制及其使用

磨针仪是专门用于磨制玻璃微吸管尖端的小型仪器。毛细玻璃管拉制成的微吸管,是生理学,神经生物学,显微手术,显微操作实验中常用的工具。出于不同的目的,对玻璃微吸管尖端的要求不同。用于生理学记录细胞膜内外电位差的微电极,和用于体细胞或卵细胞核内注射大分     

细胞膜电位研究的一个新进展——线粒体膜电位

四十年前,Hodgkin 和 Huxley 找到了一种较为满意的生物材料——枪乌贼的巨神经纤维,其直径可达1/3～1毫米,并用细胞内微电极测得了其静息电位值。由于细胞内微电极的进一步发展,其尖端直径可细到0.1～0.2毫米以及对线粒体的特殊处理可使其直径增大。近十年来细胞膜电位研究的一个新方向,就是线粒体膜电位的研究。     

微电极尖端定位的组织切片直接印相方法

在神经电生理研究中应用微电极技术时,必须对微电极尖端进行组织学定位,一般都是采用蓝点标记法,在实验结束后用组织学方法确定蓝点位置,然后照相记录。但常规显微照相手续较复杂,我们改用了一种简便易行的直接放大印相方法,介绍如下。 实验结束后,按常规要求将观察部位的神经组织取材固定,做冰冻切片,厚约40μm,铺     

选择性微电极在植物生理学研究中的应用

选择性微电极技术是一种不仅能直接测定活的生物细胞或细胞器内的离子或分子活度,而且能对活的生物相邻的位置、功能和代谢速率可能不同的特定微区细胞表面的离子或分子流（flux）分别测定的电生理方法。具有操作简便、实时、非损伤性（测定离子或分子流）、灵敏度高（可达10^-12moles cm^-2s^-1）等优点。因为它是用微型化（尖端直径为0.5-5μm）的离子或分子选择性电极直接对准样品测定,不同于其他化学测定需取样品,所以能连续测定和自动监测,具有广阔的应用前景。该文阐述了选择性微电极测定原理,总结了选择性微电极技术在植物生理学研究中的应用进展,并展望了其发展前景。     

"植物生命活动调节"的教学后记

教学后记是教师对课堂教学现象的反思、研究和小结。教师每上一堂课 ,或多或少有点得失、感想和体会 ,如果把这些及时地写下来 ,在今后的教学中引以为戒 ,不断地改进 ,就会取得事半功倍之效果。下面是关于“植物生命活动调节”这一教学内容的教学后记。1　成功点 :比喻为了验证胚芽鞘的尖端确实能产生某种物质 ,课堂教学中插播了一段实验录像 ,即燕麦实验法过程 :“用剥线钳剪去胚芽鞘的尖端 ,放在琼脂切块上。过些时候 ,移去尖端 ,再把这些琼脂切成小块 ,放在切去尖端的胚芽鞘切面的一侧 ,结果胚芽鞘也发生了生长和弯曲的现象 ,弯向放置琼…     

記录单个神經细胞放电用的负电容补偿式高输入阻抗前置放大器

为了记录单个神经细胞发放的电脉冲,需要使用尖端极细(小于1微米)的微电极,其阻抗通常高达10—100兆欧,这就要求前置放大器的输入阻抗必须达到1,000兆欧左右。一般地说,神经细胞的放电是一个很快的尖脉冲,其     

In situ, in-liquid, all-electrical detection of Salmonella typhimurium using lead titanate zirconate/gold-coated glass cantilevers at any dipping depth

Most biosensing techniques are indirect, slow, and require labeling. Even though silicon-based microcantilever sensors are sensitive and label-free, they are not suitable for in-liquid detection. More recently lead zirconate titanate (PZT) thin-film-based microcantilevers are shown to be sensitive and in situ. However, they require microfabrication and must be electrically insulated. In this study, we show that highly sensitive, in situ, Salmonella typhimurium detection can be achieved at 90% relative humidity using a lead zirconate titanate (PZT)/gold-coated glass cantilever 0.7 mm long with a non-piezoelectric 2.7 mm long gold-coated glass tip by partially dipping the gold-coated glass tip in the suspension at any depth without electrically insulating the PZT. In particular, we showed that at 90% relative humidity and with a dipping depth larger than 0.8 mm the PZT/gold-coated glass cantilever showed virtually no background resonance frequency up-shift due to water evaporation and exhibited a mass detection sensitivity of Δm/Δf = −5 × 10⁻¹¹ g/Hz. The concentration sensitivities of this PZT/gold-coated glass cantilever were 1 × 10³ and 500 cells/ml in 2 ml of liquid with a 1 and 1.5 mm dipping depth, respectively, both more than two orders of magnitude lower than the infectious dose and more than one order of magnitude lower than the detection limit of a commercial Raptor sensor.     

Studies on the origin of the tip potential of glass microelectrode

1. Tip potential (TP) of glass microelectrodes filled with 3 M KCl increased remarkably with the increase in the storage period in 3 M KCl solution at 37? C, while the electrode resistances decreased gradually.
2. The electrical conductivity through the thin glass wall near the tip was found to increase in parallel with the TP increase.
3. The e.m.f. across the thin glass wall in the tip region was directly measured. This seems to contribute to the TP generation of the microelectrode when the conductivity of the glass wall is significantly high in the tip region.
4. Effects of the acid treatment of glass employed and the acidification of fillant electrolyte solution suggested that fixed negative charges on the glass wall play a fundamental role in the TP formation.
5. Based on these experimental results, it was concluded that not only the diffusion potential through the tip pore but also the interfacial potential through the thin glass wall near the tip contributes to the TP generation, and the contribution of the latter increases with a long exposure period of the electrodes to electrolyte solution.
6. In this connection, technical problems related to reduction of the tip potential were also discussed.

     

Fabrication of glass micropipettes: a semi-automatic approach for trimming the pipette tip.

Micropipettes as research instruments are well established in cell biology, including blood rheology. However, the experimental results are, to some extent, dependent on the quality of the pipette itself; it is usually critical to have the desired pipette internal diameter and a perpendicular tip. Pipette fabrication is a two-step procedure involving: a) the pulling of the pipette from a glass capillary; b) the trimming of the pipette tip. A common method to trim and fracture the pipette tip is the use of a melted glass bead on a heated tungsten wire. Previous devices using this method were often associated with problems because the heated wire varied in length with temperature. As a result, the bead together with the attached pipette tip moved markedly and thus hampered the possibility to obtain a perpendicularly cut pipette tip. An improved design, based on the same principle with a melted glass bead, is thus suggested; it eliminates the problem with a moving glass bead and, in addition, allows semi-automatic pipette trimming by utilizing the heat-induced elongation/retraction of the heated wire to fracture the tip without requiring manual assistance. Furthermore, a simple pipette storing technique is suggested, based on standard laboratory utensils, in order to more easily handle fragile pipettes without risk of breakage.     

Tip size of ion-exchanger based K+-selective microelectrodes. I. Effects on selectivity

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to study the dependency of ion selectivity upon tip size. The selectivity of K+ ISMs depended on tip size and barrel configuration. Within the range of tip diameters tested (approximately 0.5-6 micron) all K+ ISMs constructed of two barrels glued side by side ("figure-eight glass") exhibited sensitivity to K+ and NH4+. Figure-eight K+ ISMs with tip diameters less than 1.5 micron were not sensitive to tetramethylammonium, tetraethylammonium, or choline, whereas K+ ISMs with tip diameters greater than or equal to 1.5 micron sensed all of the quaternary amines. Tip size dependent selectivity was not present in K+ ISMs made from thick septum theta glass. The explanation for tip size dependent changes in ion selectivity is unknown but a discussion of theoretical possibilities is given.     

A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

Optical oxygen sensor based on metallo-organic compound immobilized by sol-gel technique

Summary Optical sensors suitable for monitoring of O₂ in water medium were prepared by immobilization of metallo-organic complexes in xerogels created by sol-gel technique and applied to optical fibre tip or glass slides by dipping. Tris (1,10-phenanthroline)ruthenium (II) chloride was immobilized on the tip of optical fibre. The fluorescence spectra of the complex were sensitive to O₂ in water solution. The sensitivity of absorption spectra of bis(histidinato)cobalt (II) to O₂ and pH was examinated both in the solution of precursors of silica sol-gel glasses and in the complex-containing coating of glass slides.     

Electrical impedance of the labellar taste hair of the blowfly,Calliphora erythrocephala MG.

Summary The impedance characteristics (resistance and phase angle) were measured of the labellar taste hair of Calliphora erythrocephala by recording the values between the base and tip of intact and tip-amputated hairs of young and old flies.Measurements made at 30 Hz to 20 kHz indicated frequency independent values with changes in phase angle up to about 1 kHz and complete bypassing of the insulating cuticle at 15 to 20 kHz.Measurements made at 0.9 kHz with intact and amputated hairs showed a low change in phase angle and thus a negligible reactive component. Subsequently these values were treated as the resistive component of the impedance. Amputation of the tip always caused a drop in impedance of about 30 MOhm.Calculations based on known morphologic data allowed the conclusion that the measurements of intact hairs at slight dipping of the tip have been made through both channels and not the dendrite-containing channel alone; consequently the pore distad of the dendritic endings must have an electrolytic connection to both channels of the taste hair. Slightly deeper dipping of the hair tip (5 or less) resulted in 4 to 7 MOhm reduced impedance which indicates a second pore or opening in the greater tip region.This work was partially supported by National Science Foundation Grant GB-13500.     

Precise, easy measurement of glass pipet tips for microinjection or electrophysiology

We describe a simple procedure to measure precisely the tip diameter of glass micropipets. This procedure can improve the precision of microinjection and some electrophysiological measurements due to their extreme sensitivity to the pipet's exact geometric dimensions. The technique is nondestructive and readily carried out as a brief, extra step in these applications. Digital measurements of the threshold pressures for gas bubbling from (gas filled) pipets submerged in methanol were used to calculate the corresponding inner tip diameters using the LaPlace equation. Direct measurements of the inner tip diameters (from 0.2-5.0 mu) using scanning electron microscopy confirmed the validity of this indirect, simple procedure.     

A new solid-state microelectrode for measuring intracellular chloride activities.

Solid-state microelectrodes from measuring intracellular Cl activity (alphaiCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 mum), coating them under vacuum with a 0.2-0.3 mum thick layer of spectrscopic grade silver, and sealing them (except for the terminal 2-5 mum of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0+/- 0.6 m V (S,E,) per decade c hange in alphaCl. Tip resistance was (77.1+/- 3.1) x 10(9) omega(n=30). Electrode response was rapid (10-20 s), was unaffected by HCO3, H2PO4, HPO42 or protein, and remained essentially unchanged over a 24-h period. AlphaiCl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, alphaiCl significantly exceeded the value corresponding to equlibrium ditribution of Cl across the cell membrane.     

Mycoplasma attachment to solid surfaces: a review

Mycoplasma attachment to glass in a protein-containing environment requires energization of the cells, probably to provide more accessibility of binding sites. The substance mediating attachment is of protein nature. Studies with monoclonal antibodies on M. pneumoniae suggest a concentration of the binding sites at the tip structure.