ID-10-P-2528 Clearing methods for optical projection tomography microscopy

Optical projection tomography microscopy (OPT) is a modern technique that makes possible to get 3D images of specimens from 1 to 12 mm in diameter. OPT is based on acquiring sets of projections of the specimen in the range of 360° and subsequent computer reconstruction of the 3D image by a filtered back projection (FBP) algorithm. Thus the principle is similar like computed tomography, but instead of using X-ray OPT uses visible light.

        Inevitable condition for getting high quality projections is optical transparency of the specimens. Under normal circumstances fixed specimens are not transparent in majority cases. Therefore, optical clearing methods are used. Its basic principle lies in tissue dehydration and subsequent immersion in a solution that has the refractive index of proteins; thus, tissues become transparent and light does not scatter.

        Standard clearing protocol is based on dehydration of the specimen in methanol and its rinsing in, so-called, BABB solution (1 part of benzyl alcohol + 2 parts of benzyl benzoate), but BABB often washes out applied fluorescence dyes and destroys GFP signal in tissues. Thereby, a number of new clearing protocols appeared in the literature lately, namely Scale (Nat Neurosci 2011), dehydration by tetrahydrofuran and clearing by dibenzyl ether (THF+DBE; PLOS One 2012), CLARITY (Nat 2013), ClearT (Dev 2013), SeeDB (Nat Neurosci 2013).

        First, to avoid washing out the fluorescence dyes, we applied BABB on a mouse brain specimen perfunded in-vivo by tomato (Lycopersicon esculentum) lectin. In this case the staining was fixed in the tissue well, and we were successful in acquisition of inner structures, especially vessels, see Fig. 1. From practical reasons we used a block of brain of the size of approx. 3×3×3 mm³. Second, to avoid disappearance of GFP signal in mouse mutant series, we successfully applied antibodies (primary anti-rabbit, secondary donkey anti-rabbit Cy5, Abcam) against GFP to preserve signal and visualized in 3D inner structures of a young mouse heart, Fig. 2. Third, we tested Scale on a young mouse heart. Scale provides worse resulting transparency of specimens than BABB, which is documented in the literature as well, but preserves GFP signal that can be visualized in small parts of tissues, see, e.g., atria of the heart in Fig. 3. Fourth, a potentially promising method is THF+DBE. Till now we applied it to mouse embryos with only partially acceptable results. According to literature, the resulting transparency of tissues is high and should be comparable with BABB, but probably due to high amount of blood in the embryo, the resulting 3D visualization is not as clear as expected, but still we can see internal structures like a backbone, etc., Fig. 4.