High Spatiotemporal Resolution Functional Imaging of the Entire Larval Zebrafish Brain with Two-Photon Light-Sheet Fluorescence Microscopy

Light-sheet fluorescence microscopy (LSFM) has revolutionized brain imaging by enabling high-speed volumetric recording of large translucent samples, with zebrafish larvae being a prime model for whole-brain functional studies. Among its variations, two-photon (2P) LSFM stands out for its use of pulsed infrared lasers, offering deeper imaging with reduced scattering and absorption, minimal autofluorescence, and avoidance of unwanted visual responses in live animals. First introduced for functional imaging in 2011, 2P LSFM has since evolved with manifold advancements broadening its applications. Despite these advantages, challenges such as lower signal-to-noise ratios, photodamage thresholds, and the need for complex optical setups remain. In this chapter, we discuss the improvements brought by 2P LSFM and explore key innovations in this technique that address the aforementioned limitations, including optimized laser pulse frequencies, polarization techniques, and advanced beam-splitting methods to enhance fluorescence efficiency and imaging quality. We also examine the challenges associated with the generation of large datasets and introduce data processing pipelines commonly employed for efficient handling and analysis. Finally, we present a case study on the application of 2P LSFM to investigate the propagation of epileptic activity in zebrafish larvae. This approach enables real-time visualization of spatiotemporal patterns of seizure propagation across the brain, offering critical insights into the mechanisms of neuronal synchronization and circuit dysfunction. By bridging cellular and whole-brain scales, this example highlights the transformative potential of 2P LSFM in advancing our understanding of large-scale neuronal activity and complex brain disorders.