Two-Photon Bessel Beam Light-Sheet Microscopy with Time-Domain Ptychography

Two-photon (2p) fluorescence microscopy is a widely used technique for deep tissue imaging of biological samples. However, it is typically limited to confocal-type geometries, which require raster scanning and result in increased data capture times when large field of views (FOVs) are needed. To address this, we developed a 2p light-sheet fluorescence microscope technique for rapid widefield imaging. Our approach incorporates pulse compression algorithms to maintain a transform-limited pulse at the sample plane, which is critical for achieving a sufficient nonlinear photoresponse. Additionally, we use Bessel beams to ensure larger FOVs as compared to Gaussian beams [1]. Pulse compression is achieved by manipulating the spectral phase of a pulse, optimizing the 2p response. The first technique, Multiphoton Intrapulse Interference Phase Scan (MIIPS), is a phase retrieval method but is limited to quadratic phase corrections. The second technique, which is an advanced time-domain ptychography technique, i2PIE, surpasses MIIPS by reconstructing more complex spectral phases [2].