Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and biomedical applications. The spectroscopic capability of SRS enables identification and tracking of specific molecules or classes of molecules, often without labeling. SRS microscopy also has the hallmark advantage of signal strength that is directly proportional to molecular concentration, allowing for in situ quantitative analysis of chemical composition of heterogeneous samples with submicron spatial resolution and subminute temporal resolution. However, it is important to recognize that quantification through SRS microscopy requires assumptions regarding both system and sample. Such assumptions are often taken axiomatically, which may lead to erroneous conclusions without proper validation. In this review, we focus on the tacitly accepted, yet complex, quantitative aspect of SRS microscopy. We discuss the various approaches to quantitative analysis, examples of such approaches, challenges in different systems, and potential solutions. Through our examination of published literature, we conclude that a scrupulous approach to experimental design can further expand the powerful and incisive quantitative capabilities of SRS microscopy.

中文翻译：

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定量受激拉曼散射显微镜：承诺和陷阱。

自首次演示以来，受激拉曼散射 (SRS) 显微镜已成为一种强大的化学成像工具，在众多生物和生物医学应用中显示出前景。SRS 的光谱能力可以识别和跟踪特定分子或分子类别，通常无需标记。SRS 显微镜还具有信号强度与分子浓度成正比的标志性优势，允许以亚微米空间分辨率和亚分钟时间分辨率对异质样品的化学成分进行原位定量分析。然而，重要的是要认识到，通过 SRS 显微镜进行量化需要对系统和样品进行假设。这些假设通常是公理化的，如果没有适当的验证，这可能会导致错误的结论。在这篇综述中，我们重点关注 SRS 显微术中公认但复杂的定量方面。我们讨论了定量分析的各种方法、此类方法的示例、不同系统中的挑战以及潜在的解决方案。通过对已发表文献的检查，我们得出结论，严谨的实验设计方法可以进一步扩展 SRS 显微术强大而敏锐的定量能力。