Visualization of GFP mouse embryos and embryonic hearts using various tissue clearing methods and 3D imaging modalities

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, unlike the golden standard BABB protocol used for whole mount immunohistochemistry, would preserve GFP fluorescence and to compare different currently available 3D imaging modalities. We tested various 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, it caused considerable tissue shrinking artifacts when compared to the golden 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 iron from the tissues. Good 3D renderings of whole fetal hearts and embryos were obtained with OPT and SPIM, 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. Figure 1 demonstrates 3D visualizations of specimens cleared by various techniques, acquired and visualized by an OPT microscope and VolViewer software package, Bangham Lab, A)‐C) and a SPIM and Amira 3D, FEI Software, D), respectively. is an ED12.5 mouse heart with a scissor cut through the right ventricle, BABB clearing, primary antibody against GFP detected with Cy5‐coupled secondary. Red‐grey channel is transmission one, green channels is iso‐surface rendering of the fluorescent channel acquired using excitation of 628/40 nm and emission of 692/40 nm. shows an ED16.5 mouse heart with a scissor cuts through both ventricles, SCALE clearing. Maximum intensity projection (MIP) visualization of overlays of red (transmission) channel and green (fluorescent) channel using excitation of 425/40 nm and emission of 475 nm (high pass). GFP‐positive network of Purkinje fibers is clearly visible. depicts an ED12.5 mouse embryo cleared in CUBIC. MIP visualization of fluorescent channel using excitation of 425/40 nm and emission of 475 nm (high pass). GFP positivity is seen in the head arteries and heart; lot of autofluorescence is apparent in the liver. And, finally, D) represents ED12.5 mouse heart with a virtual cut through the right atrium and ventricle. Strong GFP signal (shown in red with the pseudocolor display) is visible in the pectinate muscles of the atrium and ventricular trabeculae. Cleared in CUBIC. Used abbreviations: LA – left atrium, LV – left ventricle, RA – right atrium, RV – right ventricle.