A UV LASER-SCANNING CONFOCAL MICROSCOPE FOR THE MEASUREMENT OF INTRACELLULAR CA2+

Modifications to the optics of a conventional confocal laser-scanning microscopic were made to allow imaging intracellular Ca2+-dependent fluorescence with a UV laser (351 or 364 nm). Modifications included: (1) a chromatic compensation lens in the laser path; (2) the design of a practically achromatic relay lens; (3) a longer tube length for the objective; and (4) highly reflective mirrors maximizing fluorescence measurement. This UV laser-scanning confocal microscope (UV-CLSM) yielded a lateral resolution of < 0.3 mu m and an axial resolution of < 1.5 mu m and a relevant field size of 100 mu m in diameter for a 40X objective). The effects of varying the focal length of a compensation lens, the degree of the correction for the coverglass thickness of objective and the detector aperture size on the quality of image formation were examined. Finally, UV-CLSM revealed optical sections of fine and complex structures of bullfrog sympathetic neurones loaded with a Ca2+-sensitive fluorescent probe. Changes in intracellular free Ca2+ distribution in response to high [K+] or caffeine were demonstrated. In addition, an increase in the intracellular concentration of caffeine applied externally was clearly imaged in space and time and distinguished from a resultant rise in [Ca2+](i). Thus, the UV-CLSM developed is suitable for ratiometric intracellular Ca2+ measurements and other biological studies.