Fig. 1. Energy and spectrum diagrams of CRS process. (a) Energy diagrams of CRS process, the upper part is the CARS process and the lower part is the SRL process^[14]. In CARS process, a pump photon and a Stokes photon stimulate a vibration from which a probe photon inelastically scatters. In SRL process, a pump photon and a Stokes photon excite a molecular vibration, resulting in energy transfer from the pump to the Stokes; (b) spectrum diagrams of CRS process, the upper part is the input light, and the lower part is the output light

Fig. 2. Signal composition of CARS^［17］. (a) Three components of the CARS signal plotted as a function of detuning; (b) total CARS signal, the solid line represents the sum of the contributions from Fig. (a), while the dotted line represents the nonresonant background

Fig. 3. Methods for hyperspectral CRS microscopy^[20]. (a) CRS microscopy based on frequency tuning of narrowband pump and Stokes beams; (b) CRS microscopy based on spectral focusing of broadband pump and Stokes beams; (c) multiplex CRS microscopy using a narrowband pump beam and a broadband Stokes beam

Fig. 4. Different methods for wavelength scanning. (a) Rapid wavelength scanning is performed using the slit of the pulse shaper^[33]; (b) galvanometer can control the incident angle, and the wavelength of the diffracted light in the same positon changes while the angle changes, thus rapid wavelength scanning can be performed by controlling the galvanometer^[34]; (c) rapid wavelength scanning is performed by AOTF for broadband laser^[36]

Fig. 5. Frequency-time distribution, the so-called Wigner distribution. (a) For a Fourier-transform limited laser pulse; (b) for the same laser pulse that is linearly chirped

Fig. 6. Coherent Raman excitation process^[38]. (a) Fourier-transform limited laser pulses; (b) laser pulses with the same chirp; (c) laser pulses with different chirps; (d) frequency scan with time delay

Fig. 7. Different methods for SF-CRS microscopy. (a) Introduce linear chirp through material dispersion^[56]; (b) introduce linear chirp through the spatial dispersion of the grating^[53]; (c) use AOPDF to introduce linear chirp and control time delay at the same time^[55]

Fig. 8. Different methods for multiplex CRS^[6]. (a) Signal light is spatially dispersed by the grating, and the signal is collected by the multi-channel detector; (b) signal light is dispersed in the time domain through the photon time stretching device, and the signal is collected by the high-frequency single-channel detector

Fig. 9. Raman spectra of liver, colon and adipose tissue in murine model C3H/HeN^[73]

Fig. 10. HS-SRS visualization of neutral lipid distribution in mammalian cells^[56]. (a) Mouse macrophage cells; (b) rat hepatic cells. The row to the left shows the overall cell morphology using SRS; the R_3015/2965 images in the middle row reveal predominant storage of CE in macrophage cells and TAG in hepatic cells; total lipid levels were visualized in the row to the right. The scale is 10 μm

Fig. 11. Hyperspectral SRS imaging of saturated and unsaturated fat in cancerous liver tissue^[33]. (a) Overlay image of saturated fat, unsaturated fat and protein; (b) SRS spectra; (c)‒(e) MCR reconstructed concentration maps of saturated fat, unsaturated fat and protein, respectively

Fig. 12. Typical hyperspectral SRS imaging results of a breast biopsy^[82]. (a) Multicolor SRS image of a breast core needle biopsy taken from a region shown in X-ray mammography; (b) zoomed-in areas exhibit specific regions with calcifications, densely packed cells, and connective tissue rich in lipids, proteins, collagen fibers, and hydroxyapatite; (c) SRS spectra of microcalcifications in breast tissue, standard BSA, and hydroxyapatite. Scale bars: Fig. (a) 500 μm; Fig. (b) 50 μm

Fig. 13. Hyperspectral SRS imaging and MCR analysis of human prostate cancer tissues^[85]. (a) Hyperspectral stack images acquired from 50 images at the range of 2820-3020 cm^-1, with pixel dwell time of 10 µs; (b) quantitation of lipofuscin area fraction and lipid area fraction in normal, low-grade and high-grade prostate cancer tissues; (c) student’s t test (p>0.05) of lipid area fraction (lipid area fraction is larger than 0) between low-grade and high-grade prostate cancer tissues. Scale bar is 10 µm

Fig. 14. HS-SRS microscopy reveals enrichment of drugs in living cells^[87]. SRS spectra of the bright spots in drug-treated cells match the SRS spectra of the drug in solution, but differ from that of cytosol. (a) Representative SRS images of BaF3/BCR-ABL1 cells treated with 20 mmol/L imatinib for four hours. Scale bar is 5 mm. (b) Representative SRS images of BaF3/BCR-ABL1 cells treated with 20 mmol/L nilotinib for four hours. (c) Representative SRS images of control cells treated with DMSO. (d) SRS spectra of selected ROIs in (a) and (b). (e) SRS spectra of 100 mmol/L imatinib and 100 mmol/L nilotinib solutions. (f) Comparison of average SRS spectra of cytosol of imatinib-treated, nilotinib-treated and control BaF3/BCR-ABL1 cells