| Optical imaging techniques have played important roles in monitoring of cutaneous microcirculation, tracking of tumor angiogenesis and detection of fluorescence cells, but the imaging depth and contrast is limited by the strong scattering of skin. Current skin windows provide available models, but the surgical operation always bring some side effects. The tissue optical clearing technique developed in recent years can reduce the skin scattering and enhance the light penetration depth effectively. However, relative studies were mostly based on in vitro skin. Whether this method can be applied to in vivo skin and provides a transparent window for in vivo optical imaging need to be further investigated. In this work, to image the dermal blood vessels, blood flow and cells with high resolution non-invasively, various in vivo skin optical clearing methods were developed to establish effective and safe transparent skin windows:1) Monte Carlo (MC) modeling and tissue phantom experiments for proving the improvement in optical imaging depth and contrast with optical clearing. By simulating the light distribution in tissue during skin optical clearing process with MC, we found that decrease in tissue scattering could significantly enhance the probability of photons arriving at deep tissue and improve the light penetration depth. The tissue phantom experiments verified that reduction in tissue scattering can improve the image contrast, imaging depth and sensitivity to flow velocity of Laser Speckle Contrast Imaging (LSCI) technique.2) Selection of high-efficiency skin optical clearing agents (OCAs). The collagen dissociation ability of glycerol, xylitol, sorbitol, glucose and fructose were studied with Molecular Dynamics simulation, and found that fructose and sorbitol could disrupt the hydration shell better. The results of in vitro experiments were in accordance with the results of simulation. To overcome the barrier function of stratum corneum, the enhancing effect of various penetration enhancers were investigated, and it was found that thiazone had the best enhancing effect for in vivo skin.3) Establishment of transparent dorsal skin window. Topical application of OCAs on dorsal skin could make the skin transparent within minutes, which permitted the dermal blood vessels and flow be imaged by LSCI technique with high resolution, and treatment of saline could make the skin recover to initial state quickly. Repeated application of OCAs or saline can make the transparent skin window open or close repeatedly, and the dermal blood vessels and flow were not affected.4) Footpad skin window for improving in vivo optical imaging quality. Considering the unique structure of footpad skin, high-efficiency footpad skin OCA was developed. Repeated application of the OCA or saline on footpad skin could open or close the footpad skin window repeatedly, which permitted the dermal blood vessels and flow to be monitored by LSCI technique with higher contrast and the cells be imaged by confocal microscopy with higher fluorescence intensity and imaging depth.5) Ear skin window for improving in vivo optical clearing quality. High-efficiency ear skin OCA was developed. Application of the OCA on ear skin could enhance the imaging contrast of LSCI technique greatly, and the circulating blood cells could be detected by in vivo flow cytometry with higher imaging depth.6) Safety assessment of OCAs. According to the safety evaluation method of skin topical administration, it is verified that the OCAs were non-irritant, non-allergic and non-phototoxic to skin, and they also had no toxic to organs and body. That is, these OCAs are safe to skin and body.In conclusion, transparent dorsal skin window, footpad skin window and ear skin window were established with optical clearing method in this work, which provide a safe and effective window for repeatedly imaging. These windows will be very significant for basic research and optical diagnosis, which has great scientific significance and application prospects. |
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