Figure 1: Two-photon laser-scanning microscope (TPLSM) and its point spread functions. (a) General layout of our TPLSM. Fluorescence emission was detected in nondescanned mode by photomultiplier tubes (PMTs). For ratiometric JC-1 imaging, green and red components of JC-1 emission were separated spectrally and detected by the two detection channels. (b) To estimate the spatial resolution, the point spread function of our TPLSM was determined from the intensity profiles of subresolution (100 nm) beads. Displayed intensity profiles are the averages of 24 beads, and excitation wavelength was 800 nm. Their full width at half maximum (FWHM) yields a lateral (X,Y) resolution of 0.4 μm and an axial (Z) resolution of 1.4 μm.