The microbeam facility provides a means of irradiating many cells with a specific number of ions with high spatial resolution. Exactly one (or more) particles can be targeted through the cell nucleus, through the cytoplasm, to a region outside a cell, and only to specified cells. A new Olympus inverted microscope with a motorized stage which can be computer-controlled has been acquired and installed on a new vibration isolation table. This microscope is used for off-line imaging of cells to determine their locations on the cell dish before irradiation on the microbeam facility. The stage has been modified so that it can be moved from the off-line microscope to the microbeam facility without loss of accuracy of the cell positions. This enables the locating of cells without the use of the nuclear stain which is required for on-line identification of cell nuclei. A micro-manipulator/micro-injector has been installed on this microscope to allow selection or treatment of individual cells before or after irradiation. We have also purchased a light source which can rapidly change the wavelength of the supplied light and which can be used with the off-line or on-line microscopes. The light pipe in that system to connect the light source to the microscope had such poor transmission that it was unusable. We have designed a new light pipe which will provide far higher light transmission, and it is currently being manufactured. Software development has proceeded in three different areas: 1) the addition of an irradiation protocol for the """"""""bystander"""""""" effect, 2) additional automation of repetitive tasks that do not require human judgments, and 3) integration of a new RISC based video frame grabber and processor board into the system. One of the repetitive tasks that has been automated is refocusing for each frame viewed under high magnification has been added; this requires less time than manual focusing and reduces positional uncertainty caused by poorly focused images. Integration of a new frame grabber/image processor board into the system has tripled throughput, reduced cell handling, and provided improved cellular morphometry.
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