Abstract
Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years.
Highlights
Tissue clearing using various chemicals allows visualizing the inside of large specimens as entire mouse brains or mouse embryos with modern 3Dmicroscopy, as ultramicroscopy
We introduced a resin formulation that protects samples, which are cleared with dibenzyl ether (DBE) from fluorescence fading caused by high-energy illumination and long-term storage
The refractive index of the cured resin matches the refractive index of DBE (n51.561)
Summary
Tissue clearing using various chemicals allows visualizing the inside of large specimens as entire mouse brains or mouse embryos with modern 3Dmicroscopy, as ultramicroscopy. Most of these clearing techniques rely on replacing the water content of a sample by a liquid matching the refractive index of proteins [1]. A widely used chemical tissue clearing reagent is Murray’s clearing medium (BABB) composed of one volume part benzyl alcohol (BA), and two volume parts benzyl benzoate (BB) [3]. Dibenzyl ether (DBE) is a clearing medium that provides improved tissue transparency and fluorescence preservation compared to BABB [4]. Neither BABB nor DBE prevent fluorescent samples from photobleaching due to massive light exposure, as required by PLOS ONE | DOI:10.1371/journal.pone.0114149 December 2, 2014
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