Figure 1.
Analysis by negative staining electron microscopy and immunogold labeling of the chimeric yellow fever virus/dengue virus (YFV/DENV) particles secreted in the cell supernatant.
(A): Numerous 50 to 60 nm spherical particles that had morphological characteristics of a typical flavivirus were observed by regular negative staining electron microscopy. (B): The specificity of these particles was confirmed by immunogold labeling with an anti-DENV E glycoprotein. Scale bars : 0.5 µm in A ; 100 nm in B.
Figure 2.
Analysis, by scanning electron microscopy (SEM), of Vero cells grown on microcarrier beads and infected with the chimeric YFV/DENV.
(A): At low magnification and before infection (day 0), Vero cells completely covered the microcarrier surface. (B): On day 0, analysis of these cells at high magnification showed them to have a smooth surface with protrusions. (C and D): On day 2 post-infection, several cells presented numerous chimeric viral particles at their surface (arrows). Scale bars : 100 µm in A ; 1 µm in B and C; 0.2 µm in D.
Figure 3.
Analysis, by transmission electron microcopy (TEM), of Vero cells grown on microcarrier beads and infected with the chimeric YFV/DENV.
(A and B): Ultrathin sections of the cells attached to the microcarriers showed the presence of numerous chimeric viral particles in cisterns (black arrows) linked to the rough endoplasmic reticulum. Some virus-induced smooth vesicles were observed close to rough ER cisternae containing chimeric YFV/DENV particles (white arrows). Scale bars: 0.2 µm in A and B.
Figure 4.
Principle underlying correlative scanning electron microscopy-transmission electron microscopy (CSEMTEM).
(A): SEM acquisition, at low magnification, made it possible to locate a microcarrier of particular interest (white arrow), i.e. bearing a cell with chimeric viral particles at its surface. (B): Mapping of the whole carbon disc with the multiscan module was performed, to identify the domain of interest on the disk (white circle). (C and D): After resizing (white circle in C), the sample containing the microcarrier of interest was examined under a stereo microscope (D). (E): After inclusion in Epon resin, thin sections (500 nm) of the resized sample were cut with an ultramicrotome and stained with toluidine blue, making it possible to identify the microcarrier of interest by light microscopy. (F): Ultrathin sections (70 nm) of this block were then cut for analysis of the resized sample by TEM, making it possible to visualize cells at the surface of the selected microcarrier (inset in F). The microcarrier of interest (arrows in A, D, E and F) can be followed during all these steps, through the recording of SEM, optical and TEM photographs. Scale bars : 200 µm in A, D and E; 2 mm in B and C; 100 µm in F.
Figure 5.
TEM analysis of Vero cells selected by the CSEMTEM method.
(A): Electron-dense chimeric viral particles were observed at the cell surface (black arrows). (B, C and D): Several released particles were observed at the site of small-vesicle exocytosis at the plasma membrane (white arrows). Scale bars: 0.2 µm in A, B and C; 0.1 µm in D.