Imaging pixel and frame rates are typically lower for 3- than for 2-photon excitation.
In multiphoton excitation, one pulse drives more excitation than the equivalent power distributed across multiple pulses, in the absence of fluorophore saturation (Prevedel et al., 2016; Weisenburger et al., 2019). One pulse per pixel also maximizes the acquisition rate.
Pulses per pixel across a 256 pixel line with a 4 kHz resonant galvanometer.
The rate of 3-photon image acquisition is fundamentally limited by the repetition rate of the laser. Lasers suitable for 3-photon excitation commonly have repetition rates in the 1-4 MHz range, therefore permitting acquisition from one to four million pixels per second at one pulse per pixel. For a 512 x 512 pixel image, a 1 MHz laser therefore supports 1,000,000 / (512 x 512) = 3.81 images per second with a linear galvanometer.
Faster image rates can be acquired with resonant galvanometers. With resonant galvanometers, the speed at which the laser is swept across the sample changes across the scan field, resulting in fewer pulses per pixel in the center of the field of view than toward the edges. For a 256 x 256 image acquired with a 4 kHz resonant galvanometer, 4 MHz is the minimum pulse rate required for at least one pulse at every pixel. 4 MHz lasers are available, permitting 3-photon excitation at ~32 images per second.