The Centre for Children's Burns Research, Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, QLD 4029, Australia.
Burns. 2011 Sep;37(6):1001-9. doi: 10.1016/j.burns.2011.03.013. Epub 2011 Apr 17.
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.
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，它包括在激发激光线中的非吸收频率变化。SHG 不需要任何外源性抗体或荧光标记物，并且可以从多个关键内源性生物分子的未染色切片中生成图像，适用于多种物种和不同类型的处理组织。在这里，我们在多光子显微镜（MPM）上使用 SHG 检查了正常对照的人类皮肤切片和人类烧伤疤痕组织。对正常人类皮肤和烧伤疤痕组织的检查和比较表明，真皮中的纤维排列清晰，类似于真皮胶原纤维信号。荧光染色证实 MPM-SHG 胶原与用于真皮 I 型胶原的抗体染色共定位，但与纤维连接蛋白或弹性蛋白不共定位。此外，我们能够在人类冷冻切片以及未经染色的石蜡包埋组织切片中检测到胶原的 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。这种相同的方法也成功地定位了猪和羊皮肤样本中的胶原，当可能无法获得特定于物种的抗体时，这种方法可能特别重要。总的来说，我们的结果表明，MPM SHG 检测是一种用于检查正常和受伤的人类、猪和羊真皮组织中胶原结构的高分辨率的有用工具。
