Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called ʻDurcupan™ʼ. Specimens were embedded in Durcupan™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.

中文翻译：

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一种使用新型荧光探针和稳定包埋树脂的预包埋免疫组织化学制备 CLEM 的简单方法

相关光学和电子显微镜 (CLEM) 是检查生物分子细胞定位的绝佳方法。在这里，我们开发了一种简单的 CLEM 方法，将预嵌入免疫组织化学与一种新型荧光探针 Fluolid NS Orange 和一种称为“Durcupan™”的嵌入树脂相结合。使用戊二醛和四氧化锇进行免疫标记和后固定后，将标本包埋在 Durcupan™ 或 LR White 中。接下来，在带有导航标记的取景器网格上制备超薄切片。发现嵌入 Durcupan™ 中的样品部分比嵌入 LR White 中的样品更稳定地抵抗电子束辐照。宽视场和低倍率的荧光显微镜图像和透射电子显微镜 (TEM) 图像，用相同的超薄切片独立获得。使用取景器网格条之间的三个角作为地标，将荧光显微镜图像与宽视场、低倍率 TEM 图像叠加以识别感兴趣区域，随后将其放大以确定位于荧光信号下方的细胞结构。然而，放大的 TEM 图像显得模糊，荧光信号具有模糊的外观。为了解决这个问题，放大的 TEM 图像被直接聚焦在感兴趣区域的高分辨率 TEM 图像所取代，从而促进了高分辨率 CLEM 图像的收集。