改善活体光学成像的光透明皮窗研究
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摘要
光学成像技术以极高的时空分辨率在皮肤微循环监测、皮下肿瘤血管的生成与发展、以及活体动物荧光分子探测等方面发挥重要作用,但成像深度和成像对比度往往受限于皮肤的高散射。基于外科手术的皮窗为光学成像的应用提供了一个有效的观测窗口,但术后副反应使其无法用于及时的观测。近年发展起来的组织光透明技术能有效降低组织散射、提高光在组织中的穿透深度,但现有研究多集中在离体水平。该方法能否用于活体皮肤,能否为活体光学成像提供一个可重复观测的视窗——皮窗,还有待进一步研究。
本文的工作正是针对活体成像中存在的这一问题,围绕如何建立高效、安全的光透明皮窗,改善皮下血管、血流及细胞的成像质量而展开的。主要研究内容如下：
1)理论模拟与模型实验验证光透明方法能改善光学成像深度及对比度：利用蒙特卡罗方法对皮肤光透明过程中的光分布变化进行模拟,发现组织散射的降低会增加光子到达深层组织的概率,提高光在组织中的穿透深度；通过类组织血流模型实验,证实降低组织散射能显著提高激光散斑衬比成像技术用于混浊介质中流速测量的图像对比度、成像深度和速度灵敏度。
2)高效皮肤光透明剂选型的理论与实验研究：利用分子动力学模拟对甘油、木糖醇、山梨醇、葡萄糖和果糖的胶原解离能力进行研究,发现果糖和山梨醇对胶原水化层的破坏能力更强；离体皮肤光透明实验的结果与理论模拟结果一致。为克服皮肤角质层对光透明剂的阻挡作用,比较多种化学促渗剂对在体皮肤光透明的增强作用,发现噻酮的增强效果最好。
3)可开合大鼠脊背光透明皮窗的建立：光透明剂作用于大鼠脊背皮肤表面能使皮肤在数分钟内透明,利用激光散斑衬比成像技术能够实现对皮下血管结构和血流的高分辨成像,生理盐水涂抹即可使皮肤迅速恢复至初始的混浊状态；用光透明剂局部重复处理皮肤,可实现皮下血管与血流的重复成像,且不会对皮下血管和血流造成影响,证明了基于光透明方法的大鼠脊背皮窗具有可开合的特点。
4)可开合的小鼠足垫光透明皮窗提高活体光学成像质量：针对足垫皮肤的独特结构,发展了高效的足垫皮肤光透明剂,将该光透明剂和生理盐水分别涂抹于在体小鼠足垫皮肤,能够建立一扇可开合的足垫皮窗。该皮窗不仅能够显著提高足垫皮下血管和血流的激光散斑衬比成像的对比度,也能够显著改善荧光成像的荧光信号强度和成像深度,且不会影响皮下血管和血流。
5)光透明鼠耳皮窗提高活体光学成像质量：发展了适用于鼠耳皮肤的高效光透明剂,将其局部涂抹于在体鼠耳皮肤后,能够显著提高鼠耳的光透过率,增强皮下血管结构和血流的激光散斑衬比成像的对比度；此外,光透明鼠耳皮窗还能够显著提高活体流式细胞仪对循环血细胞荧光信号的探测深度。
6)光透明剂的安全性研究：依据皮肤局部给药的安全性评价方法,证实本研究中所用光透明剂均对皮肤无刺激性、无过敏性、无光毒性,并且不会影响皮肤微结构,对器官和机体无代谢毒性。从而证明这些光透明剂对皮肤和机体是安全的。
本课题所建立的光透明皮窗(脊背皮窗、足垫皮窗和鼠耳皮窗),为活体光学成像提供了一个安全、有效的可重复观测的窗口,这对基于活体小动物光学成像的基础研究及光诊断研究至关重要,因而具有重要的科学意义与应用前景。
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.
本文的工作正是针对活体成像中存在的这一问题,围绕如何建立高效、安全的光透明皮窗,改善皮下血管、血流及细胞的成像质量而展开的。主要研究内容如下：
1)理论模拟与模型实验验证光透明方法能改善光学成像深度及对比度：利用蒙特卡罗方法对皮肤光透明过程中的光分布变化进行模拟,发现组织散射的降低会增加光子到达深层组织的概率,提高光在组织中的穿透深度；通过类组织血流模型实验,证实降低组织散射能显著提高激光散斑衬比成像技术用于混浊介质中流速测量的图像对比度、成像深度和速度灵敏度。
2)高效皮肤光透明剂选型的理论与实验研究：利用分子动力学模拟对甘油、木糖醇、山梨醇、葡萄糖和果糖的胶原解离能力进行研究,发现果糖和山梨醇对胶原水化层的破坏能力更强；离体皮肤光透明实验的结果与理论模拟结果一致。为克服皮肤角质层对光透明剂的阻挡作用,比较多种化学促渗剂对在体皮肤光透明的增强作用,发现噻酮的增强效果最好。
3)可开合大鼠脊背光透明皮窗的建立：光透明剂作用于大鼠脊背皮肤表面能使皮肤在数分钟内透明,利用激光散斑衬比成像技术能够实现对皮下血管结构和血流的高分辨成像,生理盐水涂抹即可使皮肤迅速恢复至初始的混浊状态；用光透明剂局部重复处理皮肤,可实现皮下血管与血流的重复成像,且不会对皮下血管和血流造成影响,证明了基于光透明方法的大鼠脊背皮窗具有可开合的特点。
4)可开合的小鼠足垫光透明皮窗提高活体光学成像质量：针对足垫皮肤的独特结构,发展了高效的足垫皮肤光透明剂,将该光透明剂和生理盐水分别涂抹于在体小鼠足垫皮肤,能够建立一扇可开合的足垫皮窗。该皮窗不仅能够显著提高足垫皮下血管和血流的激光散斑衬比成像的对比度,也能够显著改善荧光成像的荧光信号强度和成像深度,且不会影响皮下血管和血流。
5)光透明鼠耳皮窗提高活体光学成像质量：发展了适用于鼠耳皮肤的高效光透明剂,将其局部涂抹于在体鼠耳皮肤后,能够显著提高鼠耳的光透过率,增强皮下血管结构和血流的激光散斑衬比成像的对比度；此外,光透明鼠耳皮窗还能够显著提高活体流式细胞仪对循环血细胞荧光信号的探测深度。
6)光透明剂的安全性研究：依据皮肤局部给药的安全性评价方法,证实本研究中所用光透明剂均对皮肤无刺激性、无过敏性、无光毒性,并且不会影响皮肤微结构,对器官和机体无代谢毒性。从而证明这些光透明剂对皮肤和机体是安全的。
本课题所建立的光透明皮窗(脊背皮窗、足垫皮窗和鼠耳皮窗),为活体光学成像提供了一个安全、有效的可重复观测的窗口,这对基于活体小动物光学成像的基础研究及光诊断研究至关重要,因而具有重要的科学意义与应用前景。
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.
引文
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