Department of Biological Sciences, Carnegie Mellon University.
Department of Biological Sciences, Carnegie Mellon University;
J Vis Exp. 2023 Oct 6(200). doi: 10.3791/65338.
The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been demonstrated to be an effective and low-cost alternative to optical super-resolution microscopy. However, it has been limited by the need for specific and often custom anchoring agents to retain different biomolecule classes within the gel and by difficulties with expanding standard clinical sample formats, such as formalin-fixed paraffin-embedded tissue, especially if larger expansion factors or preserved protein epitopes are desired. Here, we describe Magnify, a new ExM method for robust expansion up to 11-fold in a wide array of tissue types. By using methacrolein as the chemical anchor between the tissue and gel, Magnify retains multiple biomolecules, such as proteins, lipids, and nucleic acids, within the gel, thus allowing the broad nanoscale imaging of tissues on conventional optical microscopes. This protocol describes best practices to ensure robust and crack-free tissue expansion, as well as tips for handling and imaging highly expanded gels.
生物样本的纳米级成像可以提高对疾病发病机制的理解。近年来，扩展显微镜（ExM）已被证明是一种有效且低成本的替代光学超分辨率显微镜的方法。然而，它受到需要特定且通常是定制的锚定剂来保留凝胶中不同生物分子类别的限制，并且还受到扩展标准临床样本格式（例如福尔马林固定石蜡包埋组织）的困难的限制，特别是如果需要更大的扩展因子或保留蛋白质表位的情况下。在这里，我们描述了 Magnify，这是一种新的 ExM 方法，可在广泛的组织类型中实现高达 11 倍的稳健扩展。通过使用甲基丙烯醛作为组织和凝胶之间的化学锚，Magnify 将多种生物分子（如蛋白质、脂质和核酸）保留在凝胶中，从而允许在传统光学显微镜上对组织进行广泛的纳米级成像。本协议描述了确保组织稳健且无裂纹扩展的最佳实践，以及处理和成像高度扩展凝胶的技巧。
