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Improved Imaging of Cleared Samples with ZEISS Lightsheet Z.1: Refractive Index on Demand - Application Note

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Application Note 2 Improved Imaging of Cleared Samples with ZEISS Lightsheet Z.1: Refractive Index on Demand Author: Mattia Carraro 1, 2 , Paul Paroutis 3 , Michael Woodside 3 and Robert V. Harrison 1,2,4 1 Auditory Science Laboratory, The Hospital for Sick Children, Toronto, ON, Canada. 2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada. 3 Imaging Facility, The Hospital for Sick Children, Toronto, ON, Canada. 4 Department of Otolaryngology – Head and Neck Surgery, University of Toronto, Toronto, ON, Canada. Date: July 2015 Compared to magnetic resonance imaging (MRI) and micro- computerized tomography (micro CT), both non-destructive imaging methodologies, the LSFM can achieve better spatial resolution (subcellular) and faster imaging speeds 2 . In addi- tion LSFM offers lower photo-bleaching and photo-toxicity, and the capability of imaging thicker tissues (>1 cm) com- pared to confocal or even two-photon microscopes 2 . Furthermore, the LSFM microscope from ZEISS, Lightsheet Z.1, can rotate the sample 360°, enabling image acquisition with different angles of view (Multiview) from which a 3D image can be reconstructed. For example using appropriate software 8,9 and mounting techniques 10 , images can be deconvolved and registered to obtain a single high-resolution 3D image file that can be viewed and interrogated with ZEN Imaging software or arivis Vision4D [arivis AG, Munich, Germany]. Light penetration in large samples (e.g., whole mouse brain tissue) is limited and requires removal of the opaque components of the sample, a technique referred to as clear- ing. Several tissue clearing protocols have been developed, all of them focused on homogenizing the refractive index (RI) within the sample and some of them based on lipid removal 11–23 . Only one published method 21 describes a variable RI, but the transparency achieved is limited and the minimum time needed to completely clear the specimen is 2 weeks. On the other hand, organic solvent based clearing approaches 11,13,14,24,25 are straightforward and rapid 26 , e.g. 3 to 5 days to produce very transparent specimens, but have some drawbacks. For example they result in decreased sig- nals from GFP-like proteins, and are corrosive for most objec- tives; most importantly their RI cannot be easily adjusted. Typically these (corrosive) organic solvents have non-adjust- able RIs between 1.53 – 1.56. Matching of the imaging solu- tion to the sample RI and the imaging objective RI is impor- tant to avoid spherical aberration. Even small mismatches in RI can produce significant decreases in images resolution and brightness 27,28 . Introduction Light Sheet Fluorescence Microscopy (LSFM) uses a thin plane of light (light sheet) to optically section trans parent tissues or whole organisms that have been fluorescently labeled. The emitted light is collected by a separate objective positioned perpendicular to the light sheet plane, allowing the imaging of the (excited) tissue during the illumination of only a single thin section of the sample. With no need for a pinhole aperture, the full optical section plane can be captured at once, providing a faster solution compared to laser scanning microscopes 1–7 .

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