Centre for Education and Research on Ageing and the ANZAC Research Institute, Sydney Medical School, University of Sydney and Concord RG Hospital, Sydney 2139, Australia.
J Struct Biol. 2010 Sep;171(3):382-8. doi: 10.1016/j.jsb.2010.06.001. Epub 2010 Jun 4.
Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.
窗孔是肝窦内皮细胞中的孔，可在血液和肝细胞之间过滤基质和碎片。窗孔在衰老和脂蛋白调节中具有重要作用。但是，其较小的尺寸（<200nm）阻止了通过明场显微镜进行任何功能分析。我们采用结构光照明显微镜，以极高的清晰度和空间分辨率观察分离的大鼠肝窦内皮细胞中的窗孔。通过该方法，阐明了窗孔（直径 123+/-24nm）和筛板的三维结构，并表明窗孔出现在细胞质突然变薄的区域（165+/-54nm 与非窗孔区域的 292+/-103nm 相比，P<0.0001）。筛板并非优先与荧光标记的 F-肌动蛋白应力纤维和内皮型一氧化氮合酶共定位，但似乎出现在细胞膜的主要衰减非筏状区域。未观察到迷宫状结构，所有窗孔均为短圆柱形孔。总之，三维结构光照明显微镜使荧光免疫染色和共定位的强大功能得以实现，从而揭示了有关窗孔和筛板的新的结构和功能信息。
