Stimulation of living cells by femtosecond near-infrared laser pulses

We have demonstrated that intracellular Ca2+ waves in a living HeLa cell can be induced by femtosecond near-infrared laser pulses. In this paper, we present the results of investigation on the process of the Ca2+ wave generation using pharmacological methods to determine generation mechanisms. A mode-locked Ti:Sapphire laser (780nm, 80fs, 82MHz) was used as a wave-triggering light source. The laser beam was focused into HeLa cells by using a water immersion objective lens (NA 0.9). Ca2+ waves were visualized by using a fluorescent Ca2+ indicator (Fluo-4) and monitored by a fluorescence microscope. Three mechanisms for the Ca2+ wave generation were considered; 1) Ca2+ flow into cells by destruction of the cell membrane, 2) mechanical stress by shock waves associated with the laser absorption, and 3) the leaking of Ca2+ through the destruction of intracellular Ca2+ stores. To investigate the mechanisms, we have performed experiments to determine the dependence of the probability of Ca2+ wave generation with two kinds of extracellular solutions;. a) a Ca2+ free extracellular solution (by use of EGTA), and b) a solution containing U-73122 to inhibit the response to shockwave-based mechanical effects. From these experimental results, we can conclude the main mechanism of Ca2+ wave, generation by laser irradiation is due to the leaking of Ca2+ through the destruction of intracellular Ca2+ stores.