Correlative light electron microscopy (CLEM) for tracking and imaging viral protein associated structures in cryo-immobilized cells
Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to...
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| Main Authors: | , , , |
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| Format: | Article (Journal) Video |
| Language: | English |
| Published: |
9 July 2018
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| In: |
JoVE. Video journal
Year: 2018, Issue: 139, Pages: 1-9 |
| ISSN: | 1940-087X |
| DOI: | 10.3791/58154 |
| Subjects: | |
| Online Access: | Verlag, Volltext: https://doi.org/10.3791/58154 Verlag, Volltext: https://www.jove.com/video/58154/correlative-light-electron-microscopy-clem-for-tracking-imaging-viral 
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| Author Notes: | Rachel Santarella-Mellwig, Uta Haselmann, Nicole L. Schieber, Paul Walther, Yannick Schwab, Claude Antony, Ralf Bartenschlager, Inés Romero-Brey |
| Summary: | Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions. |
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| Item Description: | Enthält auch eine Versuchsbeschreibung in Textform Gesehen am 17.05.2019 Wissenschaftlicher Film. Deutschland. 2018 |
| Physical Description: | Online Resource |
| ISSN: | 1940-087X |
| DOI: | 10.3791/58154 |