Study On Three-photon Imaging Technology Of In Vivo Mouse Skin In 1700nm Band

Multiphoton microscopy(MPM)as a technique with penetration depth in the order of magnitudes of millimeters and resolution of subcellular level has been widely used for in vivo and ex vivo deep biological imaging.However,due to the signal-to-background-ratio,penetration depth of 2-photon imaging is largely limited.Although scientists enhanced the imaging depth to some extent through adaptive optics or using longer excitation wavelength,it was not improved dramatically.The depth limitation of 2-photon microscopy had not been broken until Chris Xu's group at Cornell developed the technology of 3-photon imaging excited at 1700-nm window.Using this technology,they even successfully visualized neurons in mouse hippocampus without injuring the cortex.Whereas,it is still unknown that whether this advantage can be repeated on skin just like brain.Because the structure and character of skin are quite different from those of brain.For example,comparing with skin,brain tissue is relatively homogeneous and has higher transparency.Besides that,skin is highly scattering and has many layers;for instance,there are even four kinds of cellular layers 20 ?m below the surface of skin.In addition,the existence of pores and folds can also cause the refractive index mismatch,inducing the evident aberration.This is why we remain suspicious about the application of 3-photon imaging excited at 1700-nm window on skin.To address those questions above,we mainly focus our research on 5 points below:1.Developing easy-made and invasive skin window for mouse: the way people used to fix skin window on mouse is that sewing the metal wing onto mouse skin,which can lead injury to some extent.Thus,we choose to stick the metal wing onto mouse skin using dental cement instead of sewing.In this way,we can avoid leading the damage to mouse skin and satisfy the requirements for stability.2.Comparison of two different excitation wavelengths for MPM of mouse skin in vivo: to realize this contrast,we designed a two-color femtosecond laser source.Using a femtosecond laser centered at 1550 nm to pump photonics crystal rod(PC rod)and Bi Bo crystal to generate soliton at 1700-nm window and laser pulse at 800-nm window,separately.Then,utilizing this system,we proved that the excitation wavelength of 1700-nm window outperform that of 800-nm window in the term of deep tissue imaging.3.Measuring and calculating the basic parameters of different immersion medium: using the technique of “rough surface”,we precisely measured the refractive indexes of silicone oil,mineral oil and F30 CC from 400 nm to 1680 nm.Based on this result,We then fitted 8 parameters of Sellmeier equation through Origin,and acquired GVD(group velocity dispersion)as well as TOD(third order dispersion)curve,providing selection basis for choosing appropriate immersion media for MPM of skin.4.Labelling elastic fibers in mouse skin in vivo: Through orbital injection of sulforhodamine B,we successfully bypassed the stratum corneum——the barrier of skin and stained elastic fibers.After that,we combine this method and MPM excited at 1700-nm window together,and eventually broke the depth limitation of 800-nm window excitation.5.Label-free imaging of important appendant organs in mouse skin: due to the lack of intravital diagnose techniques for some essential appendant organs in skin,we tried to solve this problem in terms of imaging depth,definition and label-free.Utilizing 3-photon imaging excited at 1700-nm window,we can guarante the large imaging depth;because the difference between third order of susceptibilities of different materials can generate third harmonic generation signal,we are able to get strong signal without performing dyes.In this way,we finally realized high quality imaging of myelin sheaths and sweat ducts in mouse skin and made a solid foundation for applying this technique for human skin diagnoses.