Abstract

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast.

Highlights

  • Light-sheet fluorescence microscopy (LSFM) is rapidly becoming a landmark for neuroscience and biological processes visualization [1,2]

  • Through the first optical path, the sample is illuminated with a standard pencil-like Gaussian beam, as used in a digital scanned laser light-sheet fluorescence microscopy (DSLM)

  • We compare the results provided by the first mentioned configuration with two alternative illumination approaches allowed by the layout with the acousto-optical deflector (AOD)

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Summary

Introduction

Light-sheet fluorescence microscopy (LSFM) is rapidly becoming a landmark for neuroscience and biological processes visualization [1,2] It allows 3D imaging of biological samples with high frame rate and micron-scale spatial resolution. Thanks to these properties, LSFM has been exploited in a wide rage of applications from live imaging of fast processes up to long-term tracking of biological dynamics [3,4,5,6,7,8]. Out-of-focus contributions are avoided by exciting only the focal plane inside the sample With such intrinsic optical sectioning capability, fluorescence background is minimized, together with sample photo-damage and photo-bleaching

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