Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy
Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy
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DOI：
10.1679/aohc.67.263
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发表时间：
2004-09-01
影响因子：
--
通讯作者：
Ushiki, T
中科院分区：
文献类型：
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作者：
Kimura, E;Sekiguchi, T;Ushiki, T
阴极射线发光（CL）是从被电子束照射的材料发射的光。本研究进行了显示的适用性，生物学研究的扫描电子显微镜（SEM）配备了高灵敏度的阴极发光检测系统。为此，我们将悬浮在磷酸盐缓冲盐水中的无机荧光粉（P43）注射到大鼠血液循环中，并在一天内用戊二醛固定动物，并用扫描电子显微镜观察肝组织。我们的仪器能够同时收集这些组织的二次电子（SE）和CL图像。背散射电子（BSE）图像的同一部分也能够获得与此显微镜。SE和BSE图像清楚地显示了肝组织的三维结构，包括肝细胞，枯否细胞，伊藤细胞和窦上皮细胞，而CL图像可视化阴极发光信号从P43发射的亮点。我们在低真空条件下观察未涂覆的组织，在高真空条件下观察金属涂覆的组织，发现金属涂覆的组织的高真空观察提供了高质量的库普弗细胞中P43的CL图像。将CL图像叠加到相应的SE或BSE图像上，发现CL图像中的亮点是由枯否细胞摄取的荧光粉产生的。这些发现表明，CL以及SE或BSE信号的检测，通过SEM都为我们提供了有用的信息，在三维图像中的组织和细胞中的荧光示踪剂的分布。
Cathodoluminescence (CL) is the light that is emitted from a material irradiated by an electron beam. The present study was undertaken to show the applicability to biological studies of a scanning electron microscope (SEM) equipped with a high-sensitive cathodoluminescence detection system. For this purpose, we injected inorganic fluorescent powders (P43) suspended in phosphate buffered saline into rat blood circulation, fixed the animals with glutaraldehyde within a day, and observed the hepatic tissues with a SEM. Our instrument enabled the simultaneous collection of both secondary electron (SE) and CL images of these tissues. Backscattered electron (BSE) images of the same portion were also able to be obtained with this microscope. SE and BSE images clearly showed the three-dimensional structure of the hepatic tissues including hepatocytes, Kupffer cells, Ito cells, and sinusoidal epithelial cells, while CL images visualized cathodoluminescence signals emitted from P43 as bright spots. We observed non-coated tissues under a low-vacuum condition and metal-coated tissues under a high-vacuum condition, and found that the high-vacuum observation of metal-coated tissues provided high quality CL images of P43 in the Kupffer cells. The superimposition of the CL images onto the corresponding SE or BSE images revealed that bright spots in the CL images were produced by the fluorescent powders uptaken by Kupffer cells. These findings indicate that the detection of CL as well as SE or BSE signals by SEM all provide us with useful information on the distribution of fluorescent tracers in tissues and cells in three-dimensional images.