OnlineRepository_RawData_CLEMSIMS.zip

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

电子显微镜（Electron microscopy, EM）已沿用数十年，用于细胞结构的分析。若要同时探究特定蛋白质的定位与功能，通常需依托免疫电子显微镜（immuno-EM）技术，或是采用光镜-电镜关联显微镜（correlated light and electron microscopy, CLEM）的形式，与荧光显微镜进行关联分析。然而，上述两类方法均无法同时获取细胞的同位素组成信息。为解决这一局限，需将实验方案与二次离子质谱（secondary ion mass spectrometry, SIMS）进行关联。既往已有研究将二次离子质谱与生物样本中的电子显微镜或荧光显微镜技术进行关联，但尚未见其与CLEM结合的报道。本研究基于透射电子显微镜、常规落射荧光显微镜与纳米二次离子质谱（nanoSIMS）建立了一套实验流程，成功实现了三者的联用关联。该方案操作简便，可在高性能参数下同时运用这三项技术。我们认为，CLEM-SIMS关联技术将提供现有常规关联方法难以企及的丰富信息。