Automated Analysis of Images from Confocal Laser Scanning Microscopy Applied to Observation of Calcium Channel Subunits in Nerve Cell Model Line Subjected Electroporation and Calcium

摘要

We assess a possibility of applying automated image analysis to im-munofluorescence microphotographs from confocal laser scanning microscopy (CLSM). Several modes of automated analysis were tested to inspect differentiation of voltage dependent calcium channel subunits from a model of nerve cells PC12 (rat pheochromocytoma) subjected to electroporation (EP), with regard to extracellular calcium level. The objective of the experiments was evaluating sensitivity of the channel expression to the presence of calcium and electroporation voltage. For this purpose non-selective nanopores of a controlled conductivity were generated in the cell membrane using electroporation, at physiological or increased extracellular calcium concentrations. Introduction of Ca~(2+) into the cells was possible through electropores and physiological voltage-dependent calcium channels. Two subunits of calcium channel (α1H and α1G) were immunofluorescentically stained and the automated analysis of changes in cellular morphology was performed, based on comparative assessment of the fluorescence signal. The automated analysis allowed apparently higher observation capabilities. The results showed morphological changes in the channel subunits and higher expression of the channel, following its exposition to electric field or calcium.