title: Live-cell CLEM of subcellular targets: an optimized procedure for polymer-based imaging substrates.
abstract: Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia; Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Padman
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia; Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia.
authors.forename: Georg
authors.initials: G
authors.lastname: Ramm
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008928:Mitochondria / Q000648:ultrastructure; D011095:Polyethylenes / Q000737:chemistry; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D011095:Polyethylenes; C039184:polychlorotrifluoroethylene
keywords: Live-cell CLEM; Polymer imaging substrates; Subcellular spatial alignment; Toner transfer technique; Universal sample holder