In the field of medical research, the identification and detection of biomolecular markers are essential for the accurate diagnosis and treatment of diseases. Although fluorescence microscopy is applied commonly for single-cell analysis, many small biological molecules cannot be specifically labeled, since bulky fluorescent probes often undesirably interfere with the biological activities of biomolecules. In addition, mass spectroscopy has low spatial resolution and can also destroy tissues and cells during the detection process, rendering it unsuitable for the in situ monitoring of the temporal and spatial dynamics of biological small molecules in live cells. These limitations seriously impede the in-depth exploration of the biomolecular markers of diseases, thus emphasizing the significance of developing a powerful chemical imaging platform for the in situ research of molecules. The imaging contrast of Raman spectroscopy, which results from inelastic optical scattering based on the characteristic vibration of chemical bonds, enables it to identify chemical substances without exogenous labels. However, the low efficiency of spontaneous Raman scattering results in weak signals and time-consuming data acquisition, rendering it unsuitable in dynamic living systems. In comparison, stimulated Raman scattering (SRS) addresses the aforementioned limitations with the advantages of label free detection, high sensitivity, high chemical specificity, high-speed imaging, and high spatial resolution. It enables real-time quantitative detection of the chemical distribution and metabolic transformation of significant biological small molecules (such as lipids, amino acids, glucose, nucleic acids, collagen fibers, and monosodium urate) in live cells. The emerging technique shows broad application prospects in the field of biomedical research. This review article mainly focuses on the applications in the identification and detection of biomolecular markers of diseases, discusses the potential of advanced SRS microscopy techniques and data analysis methods to provide new pathways for precise disease diagnosis and treatment, and proposes further development trends in this research field.

SRS microscopy is an advanced label-free chemical analysis tool based on nonlinear optical processes. It has attracted considerable attention in the biomedical field. In the past few decades, in-depth studies have been conducted to improve the imaging quality of SRS microscopy, which has been widely applied to explore molecular markers. Based on the literature survey, this review introduces the latest technical advances in SRS microscopy, such as the development of various forms of hyperspectral and multiplexed SRS microscopy to improve the chemical specificity, and various bio-orthogonal small-volume Raman probes to enhance the sensitivity and specificity. With the technical progress in SRS microscopy, it has been widely used in the imaging and quantitative analysis of the molecular markers of numerous diseases, providing new insights into elucidating the pathogenesis of diseases, and showing broad prospects in disease diagnosis, process monitoring, intraoperative auxiliary detection, and subsequent treatment. This review primarily focuses on the specific biological applications of SRS microscopy in exploring the abnormal state of the biomolecular markers of atherosclerosis, gout, fatty liver and liver fibrosis, neurodegenerative diseases, infectious diseases, and cancer. Finally, this article highlights the prospects for future development in SRS microscopy.

SRS microscopy is a revolutionary imaging technique offering numerous advantages for exploring the molecular markers of diseases at the single-cell level. Owing to its advantages of being label-free, and having high speed, high sensitivity, high spatiotemporal resolution, and high chemical specificity, SRS microscopy has emerged as an ideal and promising platform for the identification and detection of biomolecular markers in diseases. We anticipate three promising directions for the SRS technique in the future. First, although the overcrowded high-wavenumber C—H stretching region (2800-3200 cm^-1) contains rich chemical information, its analysis is not comprehensive. Hence more efficient and detailed data analysis methods are required to discover molecular markers that are applicable in biomedical fields. Recent developments such as the A-PoD and penalized reference matching algorithms for SRS image processing, and the relative entropy method for Raman spectrum analysis developed by the Shi Lingyan team show broad prospects in extracting biological information and identifying molecular markers. Another limitation of SRS microscopy is its relatively low sensitivity compared with that of fluorescence microscopy; addressing this limitation can significantly promote its applications in investigating disease molecular markers. Finally, the further integration of label-free SRS imaging with multi-omics approaches is expected to provide comprehensive and accurate information about diseases. This integration enables in-depth studies on the mechanism of molecular markers in disease development, and guides disease diagnosis and treatment in clinical practice.