Two-photon light-sheet live imaging at kilohertz frame rate using birefringence-based pulse splitting

Multiphoton microscopy is widely used for imaging live and intact tissues. Its imaging speed, however, remains constrained by fluorophore emission rates and photodamage thresholds. In order to increase the effective pixel rate of a two-photon microscope beyond a few megahertz (MHz), multi-point acquisition schemes have been proposed. Two-photon (2P) light-sheet microscopy emerges as a particularly effective approach for high-speed multiphoton imaging of live specimens, as it enables parallelized excitation while minimizing the required increase in laser power. However, optimizing the signal-to-photodamage ratio in 2P light-sheet microscopy necessitates precise control over illumination parameters, including both wavelength and laser pulse repetition rate. Since conventional femtosecond laser sources generally do not allow independent modulation of these parameters, the development of low-cost, efficient, and robust strategies to modulate the temporal excitation profile is essential to fully exploit the advantages of 2P light-sheet microscopy. Here, we introduce a compact pulse-splitting scheme that meets these criteria. Our approach uses cascaded birefringent crystals to convert each excitation laser pulse into an adjustable sequence of collinear sub-pulses. We demonstrate its effectiveness in optimizing 2P light-sheet imaging of live zebrafish embryos. We analyze the impact of pulse splitting on photobleaching, nonlinear photodamage, and imaging performance. Additionally, we demonstrate high-speed 2P imaging of the beating heart and brain calcium dynamics using red fluorophores in live embryos. We achieve a kilohertz imaging frame rate, reaching more than 150 MHz pixel rates with fluorescent signal levels above 10photons⋅pixel −1 using a laser mean power and a peak intensity in the range of 100 mW and 0.1TW⋅cm −2 at the sample, respectively. This compact and adjustable pulse-splitting scheme allows full advantage to be taken of light-sheet illumination for fast in vivo 2P imaging. More generally, it facilitates the optimization of illumination parameters in multiphoton microscopy.