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Enhanced Light Sheet Elastic Scattering Microscopy by Using a Supercontinuum Laser

Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas, N. Plastira 100, 70013 Heraklion, Greece
Materials Science and Technology Department, University of Crete, 71003 Heraklion, Greece
UOC-Universitat Oberta de Catalunya, Rambla Poblenou 156, 08018 Barcelona, Spain
ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels, Spain
Vicerrectoría de Investigación, Universidad ECCI, 111311 Bogotá, Colombia
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(3), 57;
Received: 15 May 2019 / Revised: 27 June 2019 / Accepted: 2 July 2019 / Published: 5 July 2019
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
PDF [2206 KB, uploaded 5 July 2019]


Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of contrast available in every light sheet microscope. Interestingly, elastic scattering can potentially disclose valuable information from the structure and composition of the sample at different spatial scales. However, when coherent scattered light is detected with a camera sensor, a speckled intensity is generated on top of the native imaged features, compromising their visibility. In this work, we propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images: (i) polarization filtering; (ii) reducing the temporal coherence of the excitation laser light; and, (iii) reducing the spatial coherence of the light sheet. Finally, we show how these strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing, fluorescent labelling. View Full-Text
Keywords: Light Sheet Fluorescence Microscopy; biological microscopy; laser speckle; elastic scattering Light Sheet Fluorescence Microscopy; biological microscopy; laser speckle; elastic scattering

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Di Battista, D.; Merino, D.; Zacharakis, G.; Loza-Alvarez, P.; Olarte, O.E. Enhanced Light Sheet Elastic Scattering Microscopy by Using a Supercontinuum Laser. Methods Protoc. 2019, 2, 57.

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