Kaufman Jason A, Castro Monica J, Sandoval-Skeet Noemy, Al-Nakkash Layla
Department of Anatomy, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USA.
Masters of Biomedical Science Program, College of Health Sciences, Midwestern University, Glendale, AZ, USA.
J Anat. 2018 Jan;232(1):152-157. doi: 10.1111/joa.12711. Epub 2017 Oct 1.
New methods on optical clearing provide a valuable alternative to traditional physical section histology. Optical clearing allows investigation of relatively large tissue samples at histological resolution while maintaining the three-dimensional architecture of the intact system. There is significant potential for applying optical clearing to gastrointestinal tissues. In particular, intestinal crypts contain high concentrations of stem cells, making these structures especially important for research on cellular proliferation in the intestinal epithelium. The objective of our study is to demonstrate an optical clearing method that is easy to implement and is compatible with mitotic fluorescent labeling. The optical clearing method we present utilizes a Triton/DMSO delipidization step followed by refractive index matching, rendering the tissue nearly transparent. We use EdU click chemistry to fluorescently label mitotic cell nuclei. Our results demonstrate successful clearing of jejunal samples with readily visible EdU staining by means of confocal microscopy. Three-dimensional reconstruction of labeled samples reveals preservation of intestinal cytoarchitecture including muscular, submucosal, and mucosal layers. Additionally, the morphology of intestinal crypts and individual EdU-positive mitotic nuclei are visible in sharp detail within their intact three-dimensional organization. In summary, we present an optical clearing method that is easy to implement and has the potential to provide more accurate assessment of cellular proliferation within the gastrointestinal tract in both healthy and disease states.
光学透明化的新方法为传统的物理切片组织学提供了一种有价值的替代方法。光学透明化能够在保持完整系统三维结构的同时，以组织学分辨率对相对较大的组织样本进行研究。将光学透明化应用于胃肠道组织具有巨大潜力。特别是，肠隐窝含有高浓度的干细胞，这使得这些结构对于肠道上皮细胞增殖的研究尤为重要。我们研究的目的是展示一种易于实施且与有丝分裂荧光标记兼容的光学透明化方法。我们提出的光学透明化方法采用Triton/DMSO脱脂步骤，随后进行折射率匹配，使组织几乎变得透明。我们使用EdU点击化学法对有丝分裂细胞核进行荧光标记。我们的结果表明，通过共聚焦显微镜可以成功地对空肠样本进行透明化处理，并清晰地看到EdU染色。标记样本的三维重建显示，包括肌肉层、黏膜下层和黏膜层在内的肠道细胞结构得以保留。此外，在完整的三维组织结构中，可以清晰地看到肠隐窝和单个EdU阳性有丝分裂细胞核的形态。总之，我们提出了一种易于实施的光学透明化方法，该方法有可能在健康和疾病状态下更准确地评估胃肠道内的细胞增殖情况。
