PDF(Pubmed)
Abstract：
Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue.
摘要：
先进的3D高分辨率成像技术对于研究生物挑战至关重要，如神经回路分析和完整组织中的肿瘤微环境。然而,内源性荧光蛋白在清除或扩大的生物样品中发出的荧光信号随着重复照射和长时间成像逐渐减弱，损害其准确描述潜在科学问题的能力。我们已经开发了一种策略，可以在长时间的高分辨率三维成像过程中保留清除和扩大的组织样品中的荧光。我们评估了不同浓度的各种化合物，以确定它们增强荧光强度和耐光漂白性的能力，同时保持组织的结构完整性。具体来说,我们调查了EDTP利用率对GFP的影响，因为它已经被观察到显著提高荧光强度，耐光漂白，并在延长的室温储存期间保持荧光。这一突破将促进扩展的亲水性和基于水凝胶的清除和扩展方法，通过有效保护组织内的荧光蛋白，实现清除的生物组织的长期高分辨率3D成像。
