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Proceedings Paper

A new versatile clearing method for brain imaging
Author(s): Irene Costantini; Antonino Paolo Di Giovanna; Anna Letizia Allegra Mascaro; Ludovico Silvestri; Marie Caroline Müllenbroich; Leonardo Sacconi; Francesco S. Pavone
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Paper Abstract

Light scattering inside biological tissue is a limitation for large volumes imaging with microscopic resolution. Based on refractive index matching, different approaches have been developed to reduce scattering in fixed tissue. High refractive index organic solvents and water-based optical clearing agents, such as Sca/e, SeeDB and CUBIC 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 to large volumes. 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 blocks of dysplastic human brain transformed with CLARITY, immunostained and cleared with the TDE. 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.

Paper Details

Date Published: 10 March 2015
PDF: 8 pages
Proc. SPIE 9305, Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics II, 930514 (10 March 2015); doi: 10.1117/12.2076908
Show Author Affiliations
Irene Costantini, Univ. degli Studi di Firenze (Italy)
Antonino Paolo Di Giovanna, Univ. degli Studi di Firenze (Italy)
Anna Letizia Allegra Mascaro, Univ. degli Studi di Firenze (Italy)
Ludovico Silvestri, Univ. degli Studi di Firenze (Italy)
Marie Caroline Müllenbroich, Univ. degli Studi di Firenze (Italy)
Leonardo Sacconi, National Institute of Optics, Consiglio Nazionale delle Ricerche (Italy)
Univ. degli Studi di Firenze (Italy)
Francesco S. Pavone, Univ. degli Studi di Firenze (Italy)
National Institute of Optics, Consiglio Nazionale delle Ricerche (Italy)


Published in SPIE Proceedings Vol. 9305:
Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics II
Henry Hirschberg M.D.; E. Duco Jansen; Samarendra K. Mohanty; Nitish V. Thakor; Qingming Luo; Steen J. Madsen, Editor(s)

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