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.

Ricci P., Sancataldo G., Gavryusev V., Franceschini A., Mullenbroich M.C., Silvestri L., et al. (2020). Fast multi-directional DSLM for confocal detection without striping artifacts. BIOMEDICAL OPTICS EXPRESS, 11(6), 3111-3124 [10.1364/BOE.390916].

Fast multi-directional DSLM for confocal detection without striping artifacts

Sancataldo G.;Franceschini A.;Pavone F. S.
2020

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.
Ricci P., Sancataldo G., Gavryusev V., Franceschini A., Mullenbroich M.C., Silvestri L., et al. (2020). Fast multi-directional DSLM for confocal detection without striping artifacts. BIOMEDICAL OPTICS EXPRESS, 11(6), 3111-3124 [10.1364/BOE.390916].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/424230
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