【作者】 熊俊；

【导师】 瞿安连；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2007， 博士

【摘要】 细胞内外离子的动态平衡是细胞赖以生存的基础,其不平衡往往会导致疾病的发生。深入研究单个细胞内外离子活动不仅有助于了解细胞生命活动规律,也有助于揭示疾病的发病机理并促进新药的开发与筛选。应用光、电信号联合检测单细胞离子活动和其浓度变化是近年来生命科学研究的重要方法和研究热点。单细胞离子光电联合检测系统将膜片钳技术、钙离子荧光检测技术及钙离子紫外光释放技术等联合起来,并且还可以实现膜电容分泌信号和碳纤电极信号的同步测量。因而适合于在单细胞水平研究高时间分辨率的钙依赖的生理功能,为细胞生物学和膜生物物理学等领域的研究提供一种重要的工具。本文的工作主要是研究与开发一种新型的单细胞离子光电联合检测系统。主要内容如下:1.研究并实现了计算机控制膜片钳放大器的自动校准算法。在详细分析探头动态特性基础上提出了一种简化的探头传递函数,从而简化了探头放大器补偿环节的自动校准算法。实验结果表明,使用该算法能快速准确地完成探头补偿电路的校准,有效提高系统响应速度和带宽。所设计的自动校准程序模块能快速准确地获得系统相关参数,为提高膜片钳放大器测量的精度奠定基础。2.研究并实现了计算机控制膜片钳放大器的自动测试算法。自动测试模块能在4~5分钟内完成对膜片钳放大器各个功能模块的测试,包括增益、噪声和失调等性能参数,并根据测试协议判断每个模块是否工作正常,并在潜在的故障位置给出提示,帮助用户和开发人员对仪器进行测试和故障诊断。3.完成和改进了膜片钳实验参数的自动补偿算法。实现膜片钳实验过程中电极失调电位、快慢电容等参数的自动补偿,并根据实验测试过程中发现的问题对电路和算法进行了改进。4.为了克服传统膜片钳在电流钳模式下的固有缺陷,在传统膜片钳放大器中实现电压钳控制电流钳模式。重新设计了探头放大器电路,并对膜片钳放大器系统电路进行一定的改进,从而实现了钳位膜电位的同时记录瞬态膜电位变化。5.设计了基于双波长荧光显微测量原理的单细胞内游离钙离子浓度检测软件模块,分别实现系统参数校准和设置、实验刺激协议设置以及实验结果计算和显示等部分的功能。更多还原

【Abstract】 Homeostasis between intracellular and extracellular ion concentrations is of critical importance for a variety of the life functions and is tightly related to some disease. Deep study of the intracellular and extracellular ion activities of single cell will be helpful for understanding the mechanisms underlying cellular life functions as well as exploring the pathogenesis of disease and facilitating the discovery or filtration of the new drugs. Studying the ion activity and the transformation of ion concentrations in single cell by employing the optical and electrical signals detection technology becomes an important methodology and of highly interest among researchers around the world.The patch-clamp technique, technique of fluorometric measurement of intracellular Ca2+ concentration and flash photolysis of caged Ca2+ were combined to found a photoelectric associated detection system for single cellular ion activities, which also can be used to record membrane capacitance and carbon fiber electrode signals of secretion. As a consequence, the detection system is fit for studying the calcium-dependent life function in single cell with high temporal resolution and acts as a key tool in cell biology and membrane biophysics fields. This dissertation is mainly dedicated to study and develop the novel photoelectric associated detection system. It consists of following aspects:1. The automatic calibration algorithm of computer-controlled patch-clamp amplifier was studied and implemented. This thesis advanced an approximate transfer function by experience and test results. Further more, a simplified automatic regulation method of frequency compensation system in resistor-feedback patch-clamp amplifier is proposed and implemented. Experimental results demonstrate that the bandwidth of the amplifier can be substantially extended and the step response error of the headstage can be minimized after the frequency compensation system was regulated using this method. The system parameters can be obtained quickly and exactly by the automatic calibration routine. Thereby, applying the calibration routine resulted in distinct and effective improvements of the performance and accuracy of the patch-clamp amplifier.2. The automatic test algorithm of computer-controlled patch-clamp amplifier was studied and implemented. All parameters of each functional circuit in the amplifier, such as gain, noise and offset, can be obtained by automatic test routine within four to five minutes, and the operating state of each functional circuit can be evaluated according to the comparison between the test results and test protocol. The automatic test routine helps the manufactures and users for performance evaluation and troubleshooting.3. The circuit modules and algorithms used for automatic adjustment of the parameters in patch-clamp experiments were researched and improved. Based on the theoretical analysis of the parameters, the algorithm was implemented to remove the electrode offset and capacitance transient currents, and moreover, the circuits and algorithms of the automatic adjustment modules were improved according to the problems that were found in the experiments at our laboratory.4. In order to overcome the essential limitations of the conventional patch-clamp amplifier in current-clamp experiments, a new headstage was designed. Furthermore, voltage-clamp-controlled current-clamp technique was developed by design a new circuit. As a consequence, transient changes of the membrane potential can be recorded by injecting the command current into the cell while the clamp voltage is kept at a constant value.5. Base on the principle of the dual-wavelength fluorescence probe, the routines for fluorometric measurement of intracellular free calcium ion concentration were designed. The routine modules, such as system parameters calibration and setup module, experimental protocols setup module, experimental data processing and display module were implemented.更多还原