5.3.1　Two-photon confocal laser scanning microscop...

In view of the limitations of single-photon confocal fluorescence imaging technique in the optical imaging of intravital tissues，Denk et al.proposed the concept of two-photon fluorescence microscopy in 1990，which provided open ideas for optical imaging of intravital tissues.Then，the development of multiphoton fluorescence microscopy had further expanded the application of fluorescence imaging technique in clinical medicine.Compared with traditional single-photon confocal fluorescence imaging technique，the advantages of two-photon or multiphoton fluorescence imaging are as follows：①Negligible damage to biological samples prolongs effective observation time，which is convenient for long-term in vivo observation.②The long-wavelength lasers exhibits the small scattering coefficient in living tissues，deep tissue penetration，clear and high resolution fluorescence image.③The high fluorescence collection rate enhances image contrast.④The requirement for the optical path collection system is lower than that of the single-photon confocal microscope（Figure 5-5）.⑤A single wavelength of excitation light can simultaneously excite multiple dyes，so that different information in the same life activity can be obtained for mutual comparison and supplement.Therefore，multiphoton fluorescence imaging technique has been widely used in intravital tissue imaging，especially skin tissue imaging.Recently，this technique has also made breakthroughs in clinical diagnosis.The detection system based on multiphoton tomography has been applied in early skin cancer diagnosis and skin aging detection.(https://www.daowen.com)