Kanemaru Takaaki, Kondo Teruyoshi, Nakamura Kei-Ichiro, Morimoto Hiroyuki, Nishi Kentaro, Isobe Shin-Ichiro
Department of Morphology Core Unit, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
Department of Animal Pharmaceutical Sciences, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshinomachi, Nobeoka, Miyazaki 882-8508, Japan.
Microscopy (Oxf). 2021 Aug 9;70(4):368-374. doi: 10.1093/jmicro/dfab005.
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 'Durcupan™'. 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.
correlative light and electron microscopy (CLEM) 是一种用于检查生物分子细胞定位的优秀方法。在这里，我们开发了一种简单的CLEM方法，将预包埋免疫组织化学与一种新型荧光探针（即 Fluolid NS Orange）和一种名为“Durcupan™”的包埋树脂相结合。标本在使用戊二醛和四氧化锇进行免疫标记和后固定后，嵌入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™ 的标本切片比嵌入LR White 的标本对电子束照射更稳定。使用同一超薄切片独立获得宽视野、低放大倍数的荧光显微镜图像和透射电子显微镜（TEM）图像。以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野、低放大倍数的TEM图像叠加，以识别感兴趣区域，随后对其进行放大以确定荧光信号下方的细胞结构。然而，放大后的TEM图像显得模糊，荧光信号也有朦胧的外观。为了解决这个问题，将放大后的TEM图像替换为直接聚焦在感兴趣区域的高分辨率TEM图像，从而便于收集高分辨率的CLEM图像。使用耐锇的Fluolid NS Orange和抗电子束损伤的Durcupan™ 进行CLEM的简单样品处理方法，能够在亚细胞水平上确定荧光信号的精确定位。
