Development of Laser Microbeam Cell Micromanipulation and Its Bioengineering Application

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The new frontier of laser biology is being developed and a novel biotechnique of cell micromanipulation by laser microbeam is being pioneered. The system is essentially an inverted microscope with a thermostat interfaced to a He-Ne laser and a <Ar^+> laser, employed for microbeam cell irradiation. Each laser beam is guided by flexible optical fiber which can connect between several laser apparatus, a laser beam selector and then a microscope. By a laser beam selector, the desired laser is selected to be irradiated to the specimen. The phase-contrasted image of the specimen is visually observed on a TV monitor screen and registered in a video tape recorder and a frame memory for a period of time after the laser irradiation. The focal spot of the laser, through a dichromatic mirror, is superimposed on the specimen image and the focused irradiation at any desired site of the specimen is acquired by moving the mechanical stage. The laser beam is guided to the shutter controller which releases the shutter of the laser for a given period of time. The focused laser spot size of 10 <micro> m in diameter is obtainable with a 10-fold objective.Cell microsurgery such as microperforation and microdissection of living cells was performed by laser microbeam using this system. Laser irradiation on the nucleolus resulted in giant cell formation. Also the possibility of cell fusion by laser irradiation was shown.The present system of laser microbeam cell micromanipulation is probably more useful for studying the effects of laser irradiation on various type of cells than the in vivo systems. And our system has many advantages for detailed investigation of the effects of laser irradiation on the subcellular level of organella.