Department of Chemistry, Columbia University.
Department of Chemistry, Columbia University; Kavli Institute for Brain Science, Columbia University;
J Vis Exp. 2022 Apr 21(182). doi: 10.3791/63547.
Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.
在探索复杂生物系统的精细组织中，可视化大量特定的生物标志物起着至关重要的作用。因此，高度多重化的成像技术越来越受到重视。在这里，我们描述了一种新兴的高度多重化的特定蛋白质振动成像平台，通过彩虹般拉曼染料的电子预共振受激拉曼散射（epr-SRS）成像，其灵敏度可与标准免疫荧光相媲美。该方法规避了传统免疫荧光中光谱可分辨通道的限制，并提供了一种单次光学方法来以亚细胞分辨率检测组织中的多个标记物。它通常与标准组织制备兼容，包括多聚甲醛固定组织、冷冻组织和福尔马林固定石蜡包埋（FFPE）的人类组织。我们预计该平台将为生物样本的蛋白质相互作用提供更全面的图像，特别是对于厚的完整组织。本方案提供了从抗体制备到组织样品染色，再到 SRS 显微镜组装，最后到 epr-SRS 组织成像的工作流程。
