Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts

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.

Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine

“…Substantial or even complete loss of GFP fluorescence occurred in intestinal samples of CX3CR1 transgenic mice after 3DISCO, iDISCO+, uDISCO, and Visikol clearing, which is in line with previous findings in other tissues . Therefore, taken together with their destructive effects on morphology, these protocols were not further considered for testing their compatibility with immunostaining with intestinal neurochemical markers.…”

In Focus in HCB

“…Such intact-tissue analysis has been mainly applied to investigate whole brains. In this issue, Kolesová et al (2016) report the application and comparison of five tissue clearing methods for wholemount 3D imaging of intact mouse embryos and embryonic as well as adult mouse heart expressing GFP in the cardiac conduction system and the arterial tree (connexin40-GFP knock-in mice strain) or in the myocardium (Nkx2.5:GFP knock-in mice strain). Not unexpectedly, a single technique was not suitable for all applications.…”

Cell and small animal models for phenotypic drug discovery

“…An example of this is the development of light sheet microscopy25,26 and optical emission computed tomography (optical-ECT)27 that allow rapid 3D imaging of live or fixed tissue. A combination of such imaging systems with protocols for tissue clearance has been useful in deep tissue imaging in live and fixed animal models 25,26,28. Such an approach also ensures 3D visualization for rapid reconstruction and analysis of tissues, thereby providing detailed information into the pathophysiology and disease progression 27–29.…”