Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca2+]i) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400–500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca2+]i distributions with three-dimensional submicrometer resolution and 10% precision are obtained at 100-μM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca2+]i in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.
© 1994 Optical Society of AmericaFull Article | PDF Article
Stefan W. Hell, Martin Booth, Stefan Wilms, Christoph M. Schnetter, Achim K. Kirsch, Donna J. Arndt-Jovin, and Thomas M. Jovin
Opt. Lett. 23(15) 1238-1240 (1998)
Watt W. Webb
Appl. Opt. 40(24) 3969-3983 (2001)
Mattias Goksör, Jonas Enger, and Dag Hanstorp
Appl. Opt. 43(25) 4831-4837 (2004)