Abstract
Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.
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Acknowledgments
We would like to thank Ms. Alena Kvasilova and Klara Krausova for their excellent technical assistance. We are grateful to Prof. Paul Mozdziak for his kind editing of the English usage and helpful criticism. This study was supported by 13-12412S from the Czech Science Foundation, Ministry of Education PRVOUK P35/LF1/5, institutional support RVO:67985823, AMVIS LH13028 and Charles University UNCE 204013.
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Supplemental Movie 1. 360-degree rotation of a volume rendering of ED16.5 mouse heart showing expression of connexin40:eGFP in the atria and cardiac conduction system. SCALE clearing, imaging in OPT. (AVI 7039 kb)
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Supplemental Movie 2. Volume reconstruction of a whole ED12.5 mouse embryo cleared in CUBIC and imaged in OPT. (AVI 8519 kb)
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Supplemental Movie 3. (animated gif). Confocal imaging of ED10.5 atrium with 25x ScaleView objective showing single cell resolution with 1 µm voxels. The atrial cells are green and have polygonal shape without any preferential directional orientation. Red blood cells are inside the cavity. (AVI 2941 kb)
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Supplemental Movie 4 (animated gif). Multiphoton imaging of outflow tract of ED10.5 Connexin40:GFP mouse heart. Green channel outlines the endocardium transitioning to the aortic sac; red channel shows the autofluorescence of the remaining tissues. SCALE clearing, 25x ScaleView objective, 1-micron voxels. (GIF 10402 kb)
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Supplemental Movie 5. 3D confocal imaging of the adult Purkinje network. CUBIC clearing, 25x ScaleView objective, 1-micron voxels. Working myocardium in red, Purkinje fibers are labeled green by connexin40:eGFP construct. (GIF 12655 kb)
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Kolesová, H., Čapek, M., Radochová, B. et al. Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts. Histochem Cell Biol 146, 141–152 (2016). https://doi.org/10.1007/s00418-016-1441-8
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DOI: https://doi.org/10.1007/s00418-016-1441-8