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Correlative light-electron microscopy of cells containing a GFP-tagged subgenomic replicon.

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posted on 06.12.2012 by Inés Romero-Brey, Andreas Merz, Abhilash Chiramel, Ji-Young Lee, Petr Chlanda, Uta Haselman, Rachel Santarella-Mellwig, Anja Habermann, Simone Hoppe, Stephanie Kallis, Paul Walther, Claude Antony, Jacomine Krijnse-Locker, Ralf Bartenschlager

(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.

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