Comparative analysis of tissue-clearing methods for whole brain imaging of adult zebrafish using BABB, iDISCO, and ECi.

BACKGROUND

Recent advancement in macro-imaging that enables 3D visualisation of intact brain tissues overcomes the limitations of conventional 2D visualisation. To this end, various tissue-clearing techniques have been developed to make tissues transparent for 3D imaging. Although the established benzyl alcohol and benzyl benzoate (BABB) method reduces opacity and preserves fluorescence of biomarkers, its carcinogenicity and incompatibility with sensitive microscopes limit its applicability.

NEW

METHODS

This study developed an alternative tissue-clearing technique suited for the adult zebrafish brains by adopting the iDISCO protocol plus the use of ethanol with ethyl cinnamate (ECi) for evaluation of fluorescence preservation following whole brain staining of the mature dopaminergic neuron (DpN) marker [i.e., tyrosine hydroxylase (TH)], for downstream imaging.

RESULTS

The ethanol with ECi method showed enhanced signal stability and excellent fluorescence retention over time. This tissue-clearing method resulted in high-resolution and consistent clarity of the complex adult zebrafish brains, hence suitable for future 3D imaging with minimal photodamage and enhanced long-term fluorescence preservation.

COMPARISON WITH EXISTING

METHODS

When compared to the BABB tissue-clearing method, ECi demonstrated more superior tissue clearing for whole-brain imaging, preserving fluorescence signals, neuronal projections and structural integrity while minimising tissue shrinkage. Unlike BABB which quenched fluorescence and caused significant sample shrinkage, ECi maintained TH-immunoreactive cell counts and fluorescence stability up to 30 days. Its compatibility with the UltraMicroscope II allows advanced neuroimaging applications.

CONCLUSION

This ethanol with ECi method offers a safe, fast and simple alternative tissue-clearing techniques suited for high-resolution and long-term imaging of the adult zebrafish brains.