A UV laser-scanning confocal microscope for the measurement of intracellular Ca2+.
Cell Calcium
; 16(3): 205-18, 1994 Sep.
Article
em En
| MEDLINE
| ID: mdl-7828174
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cálcio
/
Microscopia Confocal
/
Microscopia Ultravioleta
Limite:
Animals
Idioma:
En
Revista:
Cell Calcium
Ano de publicação:
1994
Tipo de documento:
Article
País de afiliação:
Japão