Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser.

Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.