Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue
Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue
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DOI：
10.1679/aohc.72.101
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发表时间：
2009-09-01
影响因子：
--
通讯作者：
Naguro, Tomonori
中科院分区：
文献类型：
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作者：
Inaga, Sumire;Hirashima, Sayuri;Naguro, Tomonori
本研究介绍了一种通过铂蓝（Pt-blue）处理的低真空扫描电子显微镜（LVSEM）直接观察组织学石蜡切片的新方法。铂蓝不仅用作背散射电子 (BSE) 信号增强剂，还用作组织学特异性染色剂。在该方法中，将用于传统光学显微镜 (LM) 的大鼠舌头石蜡切片用铂蓝染色溶液 (pH 9) 在载玻片上染色，并使用 BSE 检测器在 LVSEM 中观察。在 LVSEM 下，可以在 40 倍至 10,000 倍的放大倍数下轻松进行整个切片的概览以及单个细胞和组织的三维详细观察。由于 BSE 信号的产量不同，切片中观察到的每种细胞和组织都可以清楚地区分，这取决于表面结构和对 Pt-blue 的不同亲和力。因此，根据LM和LVSEM观察到的Pt蓝染色强度，我们将细胞和组织成分大致分为三组：1）强染色（LM为深蓝色，LVSEM为最亮）组，包括上皮组织、内皮细胞和肥大细胞； 2）中等染色（浅蓝色和明亮）组，包括肌肉组织和神经组织； 3)未染色或弱染色(无色、深色)的组，包括弹性纤维和胶原纤维。我们预计该方法将有助于通过 LVSEM 以比 LM 更高的分辨率对各种组织的组织学石蜡切片进行三维直接观察。
The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x 40 to x 10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.