BABB、iDISCO和ECi对成年斑马鱼全脑成像组织清除方法的比较分析

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-06-25 DOI:10.1016/j.jneumeth.2025.110520

Naemah Md Hamzah , Siong Meng Lim , Fei Tieng Lim , Mitra Amiri Khabooshan , Jan Kaslin , Abu Bakar Abdul Majeed , Kalavathy Ramasamy

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引用次数: 0

摘要

宏观成像的最新进展使完整脑组织的3D可视化克服了传统2D可视化的局限性。为此，已经开发了各种组织清除技术，使组织透明，以便进行3D成像。虽然建立的苯甲醇和苯甲酸苄酯（BABB）方法降低了生物标志物的不透明度并保留了荧光，但其致癌性和与敏感显微镜的不相容性限制了其适用性。新方法本研究开发了一种适合于成年斑马鱼大脑的替代组织清除技术，采用iDISCO方案加上肉桂酸乙酯（ECi）乙醇，用于评估全脑成熟多巴胺能神经元（DpN）标记物（即酪氨酸羟化酶（TH））染色后的荧光保存，用于下游成像。结果经ECi法测定的乙醇具有较好的信号稳定性和较好的荧光保留率。这种组织清除方法可以获得复杂的成年斑马鱼大脑的高分辨率和一致的清晰度，因此适用于未来的3D成像，具有最小的光损伤和增强的长期荧光保存。与现有方法的比较与BABB组织清除方法相比，ECi在全脑成像中显示出更优越的组织清除，保留荧光信号，神经元投影和结构完整性，同时最大限度地减少组织收缩。与BABB猝灭荧光并导致明显的样品收缩不同，ECi维持th免疫反应细胞计数和荧光稳定性长达30天。它与UltraMicroscope II的兼容性允许先进的神经成像应用。结论乙醇- ECi方法是一种安全、快速、简便的替代组织清除技术，适用于成年斑马鱼大脑的高分辨率和长期成像。

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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.

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.