Abstract
.We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction. By synchronizing the rolling shutter of a camera with the motion of laser sheet, we can obtain a uniform plane-illuminated image far beyond the confocal range of Gaussian beam. A stable optical sectioning under a wide field of view (FOV) has been achieved for up to 20 Hz volumetric imaging of large biological specimens. PULSE method transforms the extent of plane illumination from one intrinsically limited by the short confocal range ( scale) to one defined by the motor oscillation range (mm scale). Compared to the conventional Gaussian light sheet imaging, our method greatly mitigates the compromise of axial resolution and successfully extends the FOV over 100 times. We demonstrate the applications of PULSE method by rapidly imaging cleared mouse spinal cord and live zebrafish larva at isotropic subcellular resolution.
Highlights
We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction
For a conventional focused Gaussian sheet, a conflict always exists between the beam confocal range and its axial extent, thereby causing a trade-off between high axial resolution and large field of view (FOV)
Light sheet combined with nondiffracting beams, such as Airy beam[6,12] or Bessel beam,[3,13,14] can generate a thin light sheet over a long distance, which mitigates the conflict of axial resolution and FOV
Summary
We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction. Light sheet fluorescence microscopy has become an emerging technique for three-dimensional (3-D) imaging of thick samples at high speed and low photo toxicity.[1,2,3,4,5,6,7,8,9] For a conventional focused Gaussian sheet, a conflict always exists between the beam confocal range and its axial extent, thereby causing a trade-off between high axial resolution and large field of view (FOV). We propose a propagating-path uniformly scanned light sheet excitation (PULSE) method via directly oscillating the illumination objective by a VCM.
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