Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies
Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies
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DOI：
10.1016/j.burns.2011.03.013
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发表时间：
2011-09-01
期刊：
影响因子：
2.7
通讯作者：
McMillan, James R.
中科院分区：
文献类型：
--
作者：
Chen, Alice C-H;McNeilly, Celia;McMillan, James R.
利用二次谐波产生(SHG)可以获得活细胞和组织结构的高分辨率、高对比度的三维图像，二次谐波产生(SHG)包括激励激光线路中的非吸收频率变化。SHG不需要任何外源抗体或荧光团标记，并且可以从多种物种和不同类型的加工组织中生成几个关键内源生物分子的未染色切片的图像。在这里，我们用多光子显微镜(MPM)上的倍频显微镜(MPM)观察了正常人皮肤切片和烧伤疤痕组织。对正常人皮肤和烧伤疤痕组织的检查和比较显示，真皮中纤维排列清晰，类似真皮胶原纤维信号。荧光染色证实MPM-SHG胶原与真皮I型胶原抗体染色共存，而不是纤维连接蛋白或弹性蛋白。此外，我们能够在人的冰冻切片以及未染色的石蜡包埋组织切片中检测到胶原MPM-SHG信号，然后将其与相同切片中的苏木精和伊红染色进行比较。同样的方法也成功地定位了猪和绵羊皮肤样本中的胶原蛋白，当物种特异性抗体可能不可用时，这一方法可能特别重要。总之，我们的结果表明，MPM SHG检测是一种有用的工具，可以高分辨率地检测正常人和受伤的人、猪和羊的真皮组织中的胶原结构。皇冠版权(C)2011，由爱思唯尔有限公司和ISBI出版。版权所有。
High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue. Crown Copyright (C) 2011 Published by Elsevier Ltd and ISBI. All rights reserved.