A UV laser-scanning confocal microscope for the measurement of intracellular Ca2+

Modifications to the optics of a conventional confocal laser-scanning microscope were made to allow imaging intracellular Ca(2+)-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 micron and an axial resolution of < 1.5 microns and a relevant field size of 100 microns 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 Ca(2+)-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.