Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination

Two-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600-1000 mu m with less than a 4-mu m waist when using a 10x illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and similar to 2-3-mu m axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with similar to 3 mm(3) volume per 4s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

Automated summary

The use of two-photon excitation in light-sheet microscopy is limited by its narrow excitation range, but a new illumination optics proposed in this study aims to generate longer extent Bessel beams to address this limitation. A microscope constructed with this technique allows for long-term time-lapse observations and high-speed recording with similar to 2-3-μm axial resolution, expanding the applications of two-photon excitation in light-sheet microscopy.