Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue’s biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis.

近红外 (NIR) 光谱是一种强大的分析方法，可用于快速、无损和无标记地评估生物材料。与中红外光谱相比，近红外光谱在穿透深度方面表现出色，可以进行完整的生物组织评估，尽管以降低分子特异性为代价。此外，与拉曼光谱相比，它相对安全，没有激光引起的光热损伤的风险。用于生物组织表征的典型 NIR 光谱工作流程包括样品制备、光谱采集、预处理和分析。由此产生的光谱嵌入了有关组织的生物分子、结构和功能特性的内在信息。在这里，我们通过提供在生物组织中获取 NIR 光谱、地图和图像的说明，展示了 NIR 光谱在探索和诊断应用中的分析能力。通过将该协议从结缔组织中的演示应用调整和扩展到其他生物组织，我们期望非专业用户可以执行典型的 NIR 光谱研究，以表征基础研究或临床环境中的生物组织。我们还描述了如何使用该协议对结缔组织进行探索性研究，包括区分韧带类型，无损监测工程软骨发育过程中基质形成的变化，绘制横跨牛髌骨的关节软骨蛋白多糖含量和横跨关节软骨和软骨下骨的深度区域的光谱成像。根据采集模式和实验目标，典型的探索性研究可在 6 小时内完成，包括样品制备和数据分析。