Figure 1.

TIRF workstation. The choice of the laser excitation wavelength (laser 1 = 561 nm; laser 2 = 488 nm) was computer controlled; excitation (green line) and fluorescence emission (red arrow) light paths are shown. The TIRF incident angle was adjusted by an external mirror. The microscope stage and objective lens employed piezo-positioners to control specimen position and image focus. Images were acquired with an EMCCD camera. A waveform generator set the duration, delay, and frequency of photoactivation pulses (laser 3 = 405 nm), also in TIRF mode. Separate, exchangeable filter cassettes were used for GFP and tRFP fluorescence.