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

ABSTRACT: 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 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2-3-μm 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 ~3 mm³ volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

SUBMITTER: Takanezawa S

PROVIDER: S-EPMC8137944 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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

Takanezawa Sota S Saitou Takashi T Imamura Takeshi T

Nature communications 20210520 1

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 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sh ...[more]

PMID: 34016994

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