Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity.

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 ([Ca(2equation)](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 [Ca(2+)](i) distributions with three-dimensional submicrometer resolution and 10% precision are obtained at100-µM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca(2+)](i) in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.