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Global Ca2+ wave was triggered in Cell 1 after the fs-laser exposure. (a) Ca2+ wave spreading at different time slots (left). The spreading was not radially symmetrical, and the curves of Fluo-4 (right) show the arrival of Ca2+ wave and the oscillations right after. The Ca2+ wave “jumped” over Cell 3 and arrived in Cell 2 first. Cell 3 and 4 were excited at the same time even though their distance to Cell 1 was quite different. (b) The Ca2+ wave could spread over a gap of 41 μm in a Ca2+-free environment. There was also a Ca2+ oscillation in Cell 1. The speed of Ca2+ wave from Cell 1 to Cell 3 was found to be 39.2 μm/s. Fluorescence intensity was normalized by the initial base at time 0. Arrow: position of fs laser focus. Bar: 30 μm (enhanced online).. [URL: http://dx.doi.org/10.1063/1.4707375.1]10.1063/1.4707375.1
Four types of Ca2+ oscillations. (a) Common type with Ca2+ peak amplitude decreasing and spike period increasing. (b) The amplitude of Ca2+ peaks increased along time. (c) The oscillations of surrounding cells could “feedback” to the exposed cell. (d) Ca2+ level in the neighboring cell gave a peak earlier than that in the exposed cell. Arrow: fs laser exposure event.
Different Ca2+ wave spreading from the laser exposures (50 mW, 0.2 s). (a) First exposure: only one neighboring cell was excited. (b) Second exposure: no Ca2+ wave spreading. (c) Third: a large range of Ca2+ wave spreading. (d) Fourth: a large range of Ca2+ wave spreading. (e) Fifth: only one cell was excited. (f) Sixth and seventh: no Ca2+ wave spreading or local Ca2+ rise. Time interval between laser exposures was 10 min. Arrow: laser exposure position. Bar: 20 μm.
RB2 (2 μM) blocked the Ca2+ wave spreading in HeLa cells. (a) No Ca2+ spreading in Cell 2 or local Ca2+ rise in Cell 1 was observed after the laser exposure in the presence of RB2. (b) After fs laser exposure, the basal fluorescence of Cell 1 decreased to dark. A modest fluorescence rise in Cell 2 was caused by the 473-nm laser scanning during excitation. Arrow: fs laser exposure event. Bar: 10 μm.
Multi-time laser exposures (20 mW with an exposure duration of 0.1 s) were performed to modulate the Ca2+ level of Cell 1. Ca2+ wave spreading occurred (left) only when the Ca2+ level was above a threshold (right). Arrow: fs laser exposure event. Bar: 10 μm.
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