Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy
M. Caroline Müllenbroich,M. Caroline Müllenbroich,Lapo Turrini,Ludovico Silvestri,Ludovico Silvestri,Tommaso Alterini,Ali Gheisari,Natascia Tiso,Francesco Vanzi,Francesco Vanzi,Leonardo Sacconi,Leonardo Sacconi,Francesco S. Pavone,Francesco S. Pavone,Francesco S. Pavone +14 more
TLDR
This work reports how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20- fold increase in accuracy in the detection of activity correlations in functional imaging.Abstract:
Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.read more
Citations
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Light sheet fluorescence microscopy
Ernst H. K. Stelzer,Frederic Strobl,Bo-Jui Chang,Friedrich Preusser,Stephan Preibisch,Katie McDole,Reto Fiolka +6 more
TL;DR: Light sheet fluorescence microscopy (LSFM) is a technique that uses a thin sheet of light for illumination, allowing optical sectioning of the sample as discussed by the authors, and is used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution.
Journal ArticleDOI
Advanced fluorescence microscopy for in vivo imaging of neuronal activity
Giuseppe Sancataldo,Ludovico Silvestri,Anna Letizia Allegra Mascaro,Leonardo Sacconi,Francesco S. Pavone +4 more
TL;DR: The present review aims to guide the reader through the main optical systems in the field toward future directions for in vivo microscopy applications.
Journal ArticleDOI
Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.
Irina Rakotoson,Brigitte Delhomme,Philippe Djian,Andreas A. Deeg,Maia Brunstein,Christian Seebacher,Rainer Uhl,Clément Ricard,Martin Oheim +8 more
TL;DR: A large-field 2P-spinning disk microscope that permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution is described.
Journal ArticleDOI
Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector
Vladislav Gavryusev,Giuseppe Sancataldo,Pietro Ricci,Alberto Montalbano,Chiara Fornetto,Lapo Turrini,Annunziatina Laurino,Luca Pesce,Giuseppe de Vito,Natascia Tiso,Francesco Vanzi,Ludovico Silvestri,Francesco S. Pavone +12 more
TL;DR: This work reports on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector and shows that the doubling of the imaging speed does not affect the confocal detection high contrast.
Journal ArticleDOI
Flexible Multi-Beam Light-Sheet Fluorescence Microscope for Live Imaging Without Striping Artifacts.
Giuseppe Sancataldo,Giuseppe Sancataldo,Vladislav Gavryusev,Vladislav Gavryusev,Giuseppe de Vito,Giuseppe de Vito,Lapo Turrini,Lapo Turrini,Massimiliano Locatelli,Massimiliano Locatelli,Chiara Fornetto,Chiara Fornetto,Natascia Tiso,Francesco Vanzi,Francesco Vanzi,Ludovico Silvestri,Ludovico Silvestri,Francesco S. Pavone,Francesco S. Pavone +18 more
TL;DR: The advantages provided by flexible and fast AODs in generating simultaneous angled multiple beams from a single laser beam and in fast light sheet pivoting are reported and the suppression of illumination artifacts are demonstrated.
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