Montserrat Coll Lladó1,*, Linus Manubens2, Greetje Vande Velde3, Laura Batlle-Morera2, Jim Swoger1,*
1Mesoscopic Imaging Facility, EMBL-Barcelona. C/ Dr. Aiguader 88, PRBB Building, 08003 Barcelona.
2 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST). C/ Dr. Aiguader 88, PRBB Building, 08003 Barcelona.
3 Department of Imaging and Pathology/ MoSAIC, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium.
*email@example.com / firstname.lastname@example.org
Mesoscopic imaging brings the possibility to navigate inside thick tissues and even whole organs at cellular resolution. This enables a potentially revolutionary change in the way that the examination of tissues has been done: the shift from performing histology on 2D physical slices to true 3D characterization of the sample. A new window of opportunity for histopathologists to make diagnoses from biopsies and similar samples arises from two advanced optical mesoscopic imaging techniques developed recently, Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM), also known as light sheet fluorescence microscopy (LSFM). These techniques, in particular LSFM, are able to image an intact mouse brain at high resolution, giving basic clues for understanding the complex networks forming the neuronal landscape.
However, imaging thick tissues in 3D relies on making the samples transparent and also in a series of permeabilization steps to allow labelling reagents to penetrate well inside the depth of the tissue. A growing list of tissue clearing protocols has been reported over the last years. We have been exploring different methods for chemically clearing thick tissues and organs, investigating the trade-offs between clearing capability, fluorescence preservation, and changes in sample morphological properties. I will show neural related tissues and brains chemically cleared using different clearing techniques (BABB, CUBIC, iDISCO, CLARITY), and illustrate the parameters to be considered when selecting the appropriate protocol. As examples we will dive deep inside of a fungi-infected adult mouse brain, go through the architecture of a developing mouse brain, and show some of the incipient characteristics of an early stage brain organoid.
Keywords: clearing, Optical Projection Tomography (OPT), Selective Plane Illumination Microscopy (SPIM), brain, biopsy, diagnostics, development, infection, brainoids
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