Question: 

Photolysis of caged compounds to induce cytosolic reactions became a popular method within the past 20 years . However, to probe for localized and sub-cellular signaling in a two-photon based photolysis is necessary. With femtosecond pulsed infra-red lasers in the megahertz domain, the question arises whether cage substance is depleted in the focus, at a physiological relevant time scale, due to diffusion limits.

Methodology: 

Fast confocal image is a prerequisite to record cellular reactions in the mirosecond domain.We combined two-photon uncaging and kilo-beam confocal microcopy. A Titan-Saphire laser was complemented with a pulse-picker to uncage chelated calcium in the range between 4.5 kHz to 90 MHz. The experimental approach was accompanied by mathematical modeling.

Results: 

Pulsed two-photon-excitation shows a fast upstroke in the first millisecond of pulsed laser illumination, before dropping to a constant steady state level. Pulse picking modulated the interval for the system to recover to initial state.Picking laser pulses reveals to be a promising method to reduce the average laser power while maintaining signal quality.

Conclusion: 

The acquired data as well as modeling depict a depletion of caged compounds in the focus. Uncaging is ultimately limited by diffusion between laser pulses. By prolonging this time, the deposition of laser energy in the cell, can be adapted and minimized towards a reduction of laser-induced cell-damage.