Scientific Results

A versatile new technique to clear mouse and human brain

Year: 2015

Authors: Costantini I., Di Giovanna A.P., Mascaro A.L.A., Silvestri L., M├╝llenbroich M.C., Sacconi L., Pavone F.S.

Autors Affiliation: European Laboratory for Non-linear Spectroscopy, University of Florence, via nello carrara 1, Sesto Fiorentino, Florence, 50019, Italy; National Institute of Optics, National Research Council, Largo Fermi 6, Florence, 50125, Italy; Department of Physics and Astronomy, University of Florence, Via Sansone 1, Sesto, Fiorentino, 50019, Italy

Abstract: Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2\’-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.

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KeyWords: Brain; Fluorescence; Imaging techniques; Light scattering; Mammals; Optical microscopy; Photons; Plants (botany); Skin; Tissue; Transparency, CLARITY; clearing; Light-sheet microscopies; TDE; Two photon microscopy, Refractive index
DOI: 10.1117/12.2184289