Supplementary Figure 2: Synchronized control signals for the high-speed microscope.

Schematics of the timing signals used to synchronize laser pulse emission, camera frame acquisition, the scanning cycle of the galvanometer mirror, and shuttering of the laser beam by the optical chopper, using the laser pulses as the seed clock. (a) A transistor-transistor logic (TTL) digital output from the laser has the same cycle frequency, f, as the laser repetition rate. Laser pulse emission is synchronized with the galvanometer mirror’s scanning cycle such that the scanning cycle duration, t₁, contains an integral, uniform number of laser pulses (e.g. f · t₁ is an integer). (b, c) Frame acquisition by the camera is triggered on the rising edge of a digital signal, b, and is synchronized with the triangular waveform of period t₁ used to drive the galvanometer scanning mirror, c. The period, t₁, was 2–10 ms for the biological images shown in this paper. Frame acquisition and the scanning cycle are both synchronized with laser pulse emission. For a subset of experiments (Fig. 2f–h) we acquired images at 1 kHz, by driving the scanning mirror at 500 Hz and recording separate images on the forward and backward phases of the scanning motion. (d) Mechanical movement of the scanning mirror follows the triangular command waveform with a slight temporal delay, Δt ~ 250 μs. Blue portions of the trace denote intervals during which the laser beam passes through the optical chopper (see Fig. 1a). Yellow portions denote intervals when the chopper blocks the beam. The beam passes through the chopper when the mirror is in the innermost 90% of its scanning range (marked as d₁ on the graph).(e) The optical chopper shutters the laser beam during the turnaround portion of the scanning mirror’s motion. The duration, t₂, during which the beam is blocked at each extremity of the scanning motion yields a duty cycle of 80% for the illumination of the specimen. (f) Block diagram showing the flow of synchronization signals.