Study And Design Of Photoelectric Associated Detection System For Ion Activities Of Single Cell

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.