Three-photon light-sheet fluorescence microscopy

Adrià Escobet-Montalbán, Federico M. Gasparoli, Jonathan Nylk, Pengfei Liu, Zhengyi Yang, and Kishan Dholakia

To appear in Optics Letters

bioRxiv: 10.1101/323790

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 um diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.