Correlative volume microcopy of virus-containing amphisomes enabled by fluorescence-guided focused ion beam-scanning electron microscopy
Year: 2025
Type: article
Abstract: Volume correlative light and electron microscopy (vCLEM) enables ultrastructural analysis of rare cellular events. Here, we present a protocol for targeting virus-containing amphisomes in eukaryotic cells with vCLEM. We describe steps for identifying target cells using confocal fluorescence microscopy, preparing and mounting samples for focused ion beam-scanning electron microscopy (FIB-SEM), relocating the target cells and FIB-SEM stack acquisition, and correlation of volume light and electron microscopy data for 3D modeling. This protocol is optimized for imaging adherent cultured cells. For complete details on the use and execution of this protocol, please refer to Schmidt et al. 1 • A vCLEM protocol for fluorescence-guided FIB-SEM of rare cellular events • Instructions for using gridded glass-bottom dishes to relocate cells of interest • Sample preparation steps for staining, dehydration, and resin embedding for FIB-SEM • Workflow for using mitochondria as internal landmarks for high-accuracy correlation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Volume correlative light and electron microscopy (vCLEM) enables ultrastructural analysis of rare cellular events. Here, we present a protocol for targeting virus-containing amphisomes in eukaryotic cells with vCLEM. We describe steps for identifying target cells using confocal fluorescence microscopy, preparing and mounting samples for focused ion beam-scanning electron microscopy (FIB-SEM), relocating the target cells and FIB-SEM stack acquisition, and correlation of volume light and electron microscopy data for 3D modeling. This protocol is optimized for imaging adherent cultured cells.
Source: STAR Protocols
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Funders: Spine Education and Research Institute, Pfizer, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Schweizerische Multiple Sklerose Gesellschaft, Swiss Cancer Research Foundation, Swiss Vaccine Research Institute, Roman, Marga und Mareille Sobek Stiftung, Staatssekretariat für Bildung, Forschung und Innovation
OpenAlex ID: W4413270539