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Comparison of Different Tissue Clearing Methods and 3D Imaging Techniques for Visualization of GFP‐expressing Mouse Embryos and Embryonic Hearts
Author(s) -
Kolesova Hana,
Capek Martin,
Janacek Jiri,
Sedmera David
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb28
Subject(s) - green fluorescent protein , fluorescence , fluorescence microscope , fluorescence lifetime imaging microscopy , confocal microscopy , embryonic stem cell , biomedical engineering , microscopy , confocal , anatomy , chemistry , pathology , biology , microbiology and biotechnology , medicine , optics , biochemistry , physics , gene
Aims To find a suitable tissue clearing protocol preserving GFP fluorescence for whole mount imaging of embryonic and adult hearts of transgenic mice. Methods We tested various published organic solvent‐based or water‐based clearing protocols claiming to preserve GFP fluorescence in central nervous system: tetrahydrofurane dehydration and dibenzyl ether protocol (DBE), SCALE, CLARITY, and CUBIC to evaluate their use for embryonic heart tissue. Tissue transparency and preservation of GFP fluorescence was evaluated in hearts of Cx40:GFP knock in mice at stages: ED10.5 – ED18.5, and adults. We also tested several imaging techniques and evaluated their use for embryonic hearts visualization. We compared an Upright Single Photon Confocal Microscope (Olympus) with 4×, 10×, and 25× objectives after mounting the specimens into cavity slides or custom imaging chambers with an Optical Projection Tomography (OPT, custom made) and with a Single Plane Ilumination Microscopy (SPIM) (constructed according to the OpenSPIM project). Results Despite careful control of all critical parameters, DBE clearing protocol did not preserve GFP fluorescence; in addition, it caused considerable tissue shrinking and deformation. The SCALE clearing resulted in good tissue transparency up to ED12.5; at later stages and in the adults the useful depth of imaging was limited by tissue light scattering. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The CUBIC method resulted in very well cleared specimens even at the adult stages, and GFP fluorescence was also preserved. In addition, it decolorized the blood and myocardium by removing iron from the tissues. High‐resolution images were thus obtained even from deep tissue layers with a confocal microscope with a Scale View immersion 25× objective. Simultaneously, intact specimens were imaged with OPT and SPIM that have lower resolution, but are more suitable for observing large samples. Conclusions From the tested methods, the CUBIC protocol turned out to be the best for whole mount GFP cardiac specimens. SCALE technique is also suitable for younger embryos. Confocal microscopy is needed for detailed view, while OPT and SPIM enable to observe larger samples. These protocols will be used for three dimensional reconstructions of normal and abnormal specimens to visualize the developing cardiac conduction system and coronary vasculature. Support or Funding Information Supported by 13‐12412S from the Czech Science Foundation, Ministry of Education PRVOUK P35/LF1/5, institutional support RVO:67985823, and Charles University UNCE.