A New Auto-micromanipulation System for Three Dimensional Microinjection Assisted by a Confocal Laser Scanning Microscope.

Abstract

We developed a novel auto-micromanipulation system for microinjection of a low molecular weight fluorescent probe into a target cell to analyze gap junctional intercellular communication in a three-dimensional perspective in combination with vital staining of cell membranes. This computerized system consisted of an ultrasonic linear motor-driven manipulator (OMMS) which could be precisely controlled along 4 axes and a confocal laser scanning microscope (CLSM) having excitation waves of 488nm and 515nm plus a specimen stage equipped with a piezotranslator that could be accurately leveled up or down. After vital staining with PKH26, a target cell in cultured neonatal rat cardiac myocytes overlapping each other was microinjected with Lucifer Yellow or fluo-3 to evaluate whether this system was practical. The results revealed that the three dimensional coordinates of the target cell were recognizable after vital staining and the fluorescent probe microinjected into the target cell with this system portrayed reliable distribution. This system is therefore useful for the analysis of intercellular communication in three-dimensional cells and organ structures.