Biophys J, October 1999, p. 2226-2236, Vol. 77, No. 4

Ca²⁺ Fluorescence Imaging with Pico- and Femtosecond Two-Photon Excitation: Signal and Photodamage

 ^*Department for Cell Physiology, Max-Planck Institute for Medical Research, Heidelberg, Germany and  ^#Botanical Institute, Ludwig-Maximilians University, München, Germany, and  ^§High Resolution Optical Microscopy Group, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca²⁺-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca²⁺-imaging is carried out with femto- or picosecond laser pulses.

Biophys J, October 1999, p. 2226-2236, Vol. 77, No. 4
© 1999 by the Biophysical Society   0006-3495/99/10/2226/11  $2.00

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