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

A new paper from the LOB microscopy unit on high-speed multiphoton light-sheet imaging has been published in Optica. We achieved kilohertz frame rates by enhancing the signal-to-photodamage ratio during live imaging.

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

https://doi.org/10.1364/OPTICA.588084 

Lei Zhu, Dale Gottlieb, Vincent Maioli, Antoine Hubert, Frédéric Druon, Pierre Mahou, Emmanuel Beaurepaire, and Willy Supatto 

Such performance requires to mitigate nonlinear photodamage by optimizing the laser pulse temporal profile. In addition, the laser wavelength must be tuned to enhance fluorescence signal while reducing heating from water absorption. Decoupling wavelength and pulse repetition rate tunability is a challenge with conventional femtosecond lasers. Hence, the development of efficient and robust strategies to modulate the temporal excitation profile is essential to fully exploit the advantages of multiphoton light-sheet microscopy. To overcome this bottleneck, we developed a straightforward pulse-splitting strategy, inspired by the use of birefringent crystals in divided-pulse amplification (collaboration with F. Druon, Laboratoire Charles Fabry, CNRS, Institut d’Optique Graduate School, Université Paris-Saclay). We achieved 150 MHz pixel rates with low mean power, low peak intensity, and low energy per pulse. These illumination parameters are critical for minimizing sample heating, nonlinear photodamage, fluorophore photobleaching, and saturation during high-speed live imaging with multiphoton microscopy.

Congrats to all authors @LeiZHU Dale Gottlieb Vincent Maioli Antoine Hubert Frederic Druon Pierre Mahou Emmanuel Beaurepaire and Willy Supatto.