Oblique scanning 2-photon light-sheet fluorescence microscopy for rapid volumetric imaging.

ABSTRACT

Light-sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co-aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2-photon LSFM (OS-2P-LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning-descanning system. This system also provides improved light-sheet confinement against scattering by using a 2-photon Bessel beam. The OS-2P-LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high-speed, translation-free and scattering-robust 3D imaging of large biological specimens.