New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography

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• 作者：Martin Schorb^a ; ^b ; Leander Gaechter^c ; Ori Avinoam^a ; Frank Sieckmann^d ; Mairi Clarke^a ; Cecilia Bebeacua^a ; ^e ; Yury S. Bykov^a ; Andreas F.-P. Sonnen^a ; ^f ; Reinhard Lihl^g ; John A.G. Briggs^a ; ^e ; ^f ; ^{john.briggs@embl.de}
• 关键词：CLEM ; correlative light and electron microscopy ; CEMOVIS ; cryo-electron microscopy of vitrified sections ; EM ; electron microscopy ; ET ; electron tomography ; FM ; fluorescence microscopy ; FOV ; field of view ; LN ; liquid nitrogen ; NA ; numerical aperture ; POI ; position(s) of interest ; TEM ; transmission electron microscopy ; WD ; working distance
• 刊名：Journal of Structural Biology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：197
• 期：2
• 页码：83-93
• 全文大小：4230 K
• 卷排序：197

文摘

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.