Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.; van Kuppeveld, F. J. M.

Volume 1 Issue 4 e00104- 16 msphere.asm.org 14 sequentially at 5-min intervals. For the generation of movies, live-cell imaging data were processed in ImageJ and aligned using the StackReg plugin. Correlative light electron microscopy. Asymmetrical guide marks were scratched into glass-bottom eight-well chamber -slides (Ibidi); BGM(GFPS1-10) cells were grown to subconfluency and infected with CVB3-3A(S11aa2) on these slides. Just prior to imaging and to later aid in the correlation of fluorescent and EM images, cells were incubated with 100 nM MitoTracker deep red FM (Thermo Fisher) for 30 min, then washed several times in Fluorobrite medium supplemented with 8% fetal calf serum (FCS) and 25 mM HEPES. Imaging was carried out from~2 h p.i. in a live-cell chamber at 37°C and 5% CO 2 with a Leica SP8 confocal microscope equipped with a HyD detector and with a 63ϫ (1.4-NA) oil immersion objective. For each position of interest, a low-resolution tile scan was taken of the surrounding area, including guide marks, to aid in later reidentification. Z-stacks were acquired using a 1-AU pinhole and Nyquist frequency sampling. The cells were then fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Sørenson's phosphate buffer (PB) and processed for electron microscopy. The cells were postfixed first with 1% osmium tetroxide for 1 h in PB and then with 1% tannic acid in PB for 30 min. The cells were then serially dehydrated in ethanol and infiltrated and embedded in LX 112 resin (Ladd Research Industries) before polymerization at 60°C. Positions of interest imaged earlier were identified on the resin block surface through comparison with (horizontally flipped) tile scan images, the appropriate block faces were trimmed, and serial thin sections were cut and collected on copper slot grids covered with a carbon-coated formvar layer. After poststaining grids with lead citrate and uranyl acetate, image meshes spanning large areas of the grid were collected on an FEI Tecnai 20 FEG electron microscope operated at 120 kV with a charge-coupled-device (CCD) camera (US4000; Gatan) (4,000 [4K] by 4K pixel) with binning 2 and a final image pixel size of 1.94 nm. These individual images were later combined into composite stitches (65) . Cells of interest were digitally extracted from their raw image files using ImageJ and Aperio Imagescope software packages for light microscope and electron microscope images, respectively. These images were then overlaid using Adobe Photoshop CS6. The mitochondrial pattern in the EM images was compared with the MitoTracker deep red FM signal to find the right transformations to correlate both types of data. While the discrepancy in the axial resolution of the light and electron microscopes limited the precision of the overlay, the use of this secondary marker was critical to determine the z-plane in the confocal data, the best fit of the EM cell section under analysis, and the xy orientation of both images, thus providing an independent marker for an unbiased localization of the 3A signal.