Fig. 1. Development history of nonlinear optical microscopes

Fig. 2. Energy level diagrams of nonlinear effect. (a) Two photon excitation fluorescence; (b) three photon excitation fluorescence; (c) second harmonic generation; (d) third harmonic generation; (e) coherent anti-Stokes scattering; (f) stimulated Raman scattering

Fig. 3. Multiphoton and second harmonic optical microscopy^[54]. (a) Schematic diagram of optical system (AOM: acousto-optic modulator; GM: galvanometer; SL and TL: scan lens and tube lens; DM: dichroscope; M: reflector; MO: microscope objective; FL: focusing lens; F: filter; PMT: photomultiplier tube); (b) THG, SHG, and 2PEF signals of lung tumor tissue are detected by wavelength separation method with appropriate filters and merged into a THG/SHG/2PEF multi-modal image

Fig. 4. Multimodal optical imaging microscopy is used to analyze the primary and metastatic tumor microenvironment^[64]. (a) Schematic diagram of the optical imaging system based on MPEF/SHG/THG and single photon FLIM (AOM: acousto-optic modulator; IF: IR-cut filter; DM: dichroic mirror; PH: pin hole; HP: half-wave plate; FL: focal lens; FP: fiber port; PS: polarization splitter); (b) label-free imaging of metastatic tumors; (c) 3PEF image of NADH; (d) FLIM image; (e) SHG image of collagen; (f) TPEF image of FAD

Fig. 5. Multi-mode coupling of MPEF, SHG/THG, and CARS in nonlinear optical imaging systems. (a) Multimodal nonlinear optical imaging system that integrates TPEF, SHG, and CARS (PMT: photomultiplier)^[81]; (b) multimodal nonlinear optical imaging system that integrates TPEF, 3PEF, SHG, THG, and CARS (SCG: supercontinuum generation; APS: arbitrary pulse shaping)^[82]

Fig. 6. Multi-parameter optical imaging system that integrates TPEF, SHG, SRS, and TP-FLIM^[102]. (a) System schematic diagram(CO: condenser; DAQ: data acquisition system; DM: dichroic mirror; EOM: electro-optic modulator; GR: glass rod; λ/2: half-wave plate; OB: objective; PBS: polarizing beam splitter; PD: photodiode; λ/4: quarter-wave plate); (b) spatial resolution, (c) spectral resolution, and (d) temporal resolution of system

Fig. 7. High throughput nonlinear optical imaging system based on high-speed scanner. (a) Schematic diagram of a nonlinear optical microscopy imaging system based on a polygonal scanner^[112]; (b) schematic diagram of a nonlinear optical microscopy imaging system based on an acoustooptic deflector^[113]; (c) schematic diagram of a three-dimensional nonlinear optical volume imaging system based on an acoustooptic deflector and a flow detection mirror^[114]

Fig. 8. Scanning mechanism of a nonlinear optical endoscope^[181]. (a) Near end scanning combined with fiber bundle conduction; (b) optical fiber tip scanning is achieved through mechanical resonance of piezoelectric actuators; (c) MEMS systems are used for remote scanning

Fig. 9. Several common multi-parameter nonlinear endoscopic imaging systems. (a) Near-end scanning multimodal optical endoscope based on fiber bundles and multimode fibers^[170]; (b) remote scanning multimodal optical endoscope based on hollow photonic crystal double clad fiber and piezoelectric actuator^[131]; (c) multimodal nonlinear optical endoscopic imaging system based on dual core double clad fiber and micro focusing objective^[167]

Fig. 10. TPEF and SHG image analysis methods

Fig. 11. TPEF and SHG images of normal ovarian (A, D, G, J) and ovarian cancer (B, E, H, K) tissues,and SAAID index (C), TACS statistics (F), anisotropic histogram (I), and spatial correlation curves (L) of normal ovarian and ovarian cancer tissues^[185]

Fig. 12. Multivariate curve resolution for SRS image analysis

Fig. 13. TP-FLIM phasor diagram analysis

Fig. 14. Single component and multi-component fluorescence lifetime phasor plots^[197]. (a) Single component; (b) multi-component

Table 1. Characteristics of nonlinear optical microscopes