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.
Kuba
K
K
Department of Physiology, Saga Medical School, Japan.
Hua
S Y
SY
Hayashi
T
T
D000818:Animals; D002118:Calcium / Q000032:analysis*; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D008860:Microscopy, Ultraviolet / Q000295:instrumentation* / Q000379:methods*; D009474:Neurons / Q000378:metabolism; D011892:Rana catesbeiana; D013564:Sympathetic Nervous System / Q000378:metabolism
D002118:Calcium