Fig. 1. Principle and experimental program for third harmonic generation (THG) microscopy. (a) Schematic diagram of THG and spontaneous fluorescence energy levels; (b) schematic diagram of the working bands of THG and multiphoton fluorescence signals; (c) a schematic showing whether THG signal can be emitted out in different parts of two media; (d) schematic diagram of THG microscopy experimental system (GS: galvo scanning; SL: scan lens; TL: tube lens; DM: dichroic mirror; OBJ: objective lens; IF: interference filter; L: lens)

Fig. 2. Normal human brain tissue and gliomas images using THG (green) and SHG (red) imaging, the overlap between them is yellow. (a) Normal gray matter of the brain; (b) normal white matter of the brain; (c) low-grade diffuse gliomas (WHO grade II); (d) high-grade diffuse gliomas (WHO grade IV)

Fig. 3. Human healthy lung tissue and lung cancer tissue were imaged with THG (green), SHG (red), and 2PEF (blue) signal, and the other colors in the pictures are superimposed on these three colors^[51]. (a) Fresh healthy lung tissue with collagen, elastin and macrophages; (b) fresh epithelioid mesothelioma tissue, taken from bronchial biopsy, showed abnormal tumor cells and a small amount of collagen

Fig. 4. Application example of compact portable intraoperative real-time diagnostic equipment. During the operation, a portion of tissue is resected, the upper cover is opened to place a sample into the instrument, and then THG/SHG/2PEF imaging is performed to diagnose whether the tissue is normal or tumor. If it were a tumor tissue, the type and the grade would be diagnosed. This can instruct the doctor to continue the operation (drawing from the portable compact higher harmonic microscopy system FD1070^[51] developed by Professor Groot's team at VUA in the Netherlands)

Fig. 5. Schematic diagram of the endoscopic imaging principle of handheld fiber optic probes using nonlinear optical microscopy technology^[68] (redrawn schematic diagram of a handheld fast fiber optic endoscope designed by the Li Xingde's team at Hopkins University in the United States in 2018)