The spatial resolution of the Ca2+ photolysis approach permits evoking ASCs from single varicosities. Light intensity distribution of the laser spot. The laser beam was reflected back to the camera with a planar mirror placed at the level of the slice chamber and recorded with the EM-CCD camera. Stack of images made by changing the focus at 0.1-µm intervals. (A and B) The XY and YZ projections of the laser spot, respectively (bars, 2 µm). The bottom graphs show the intensity profile of the spot measured on the x axis (A) and on the z axis (B) at the level of the intensity peak (1/e2 diameter for the x axis: 1.4 µm and for the z axis: 7.2 µm). (C) Stack of epifluorescence images of an Alexa Fluor–filled MLI (left) and detail of a section of the axon (right). The photolysis positions are marked with arrowheads and numbers. (D) Individual traces recorded while sequentially stimulating the positions indicated in C. Vertical dotted line indicates the timing of the laser pulse. (E) Amplitudes (normalized in 1-µm bins to the response evoked on the varicosity center) as a function of the distance between the stimulated position and the center of the varicosity (for this analysis, only the positions located on the axon were considered). Gray circles correspond to the amplitudes from individual sweeps. Continuous line shows the fit with a Gaussian function, with a half-width of 2.18 ± 0.28 µm (mean ± SEM; n = 6 sites).
