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.