Transmission Electron Microscopy
Overview
Transmission electron microscopy (TEM) resolves subcellular structure at nanometre scale by passing electrons through ultrathin sections of fixed, heavy-metal-stained cells. In this wiki it appears for one purpose: establishing where a virus particle physically is inside a cell — specifically, whether dengue virions reach the endosomal compartment where TLR7 resides.
That is a load-bearing question for the wiki’s mechanism. TLR7 is endosomal, so any claim that dengue engages TLR7 requires the virus to get there. TEM is what supplies the direct visual evidence, and its limitations bound how strong that evidence is.
Key Points from Literature
- Synchronised-entry protocol. Virus adsorbed to isolated cells at 4 °C for 1 h with rocking (MOI 10) to allow binding without entry, then warmed to 37 °C for 5 min and immediately fixed. The cold pre-incubation is what makes the timepoint interpretable: without it, particles at different entry stages are mixed and a 5-minute snapshot means nothing (see Wang2006 - Flavivirus Activation of pDCs and TLR7 Signaling, human Plasmacytoid Dendritic Cells).
- Preparation: fixed in fresh 2.5% glutaraldehyde for 20 min, washed twice, returned to 2.5% glutaraldehyde overnight at 4 °C; post-fixed in 1% osmium tetroxide; cell block ultrathin-sectioned at 70 nm on a Reichert-Jung Ultracut E with a diamond knife; sections collected on copper grids, contrasted with lead citrate and uranyl acetate; examined on a Philips CM 10 at 80 kV (see Wang2006 - Flavivirus Activation of pDCs and TLR7 Signaling).
- What it showed: enveloped dengue-2 particles in cytoplasm, small endocytic vesicles and large endocytic vacuoles, closely associated with the Golgi apparatus and smooth ER, with the large vacuoles displaying Golgi/smooth-ER membrane characteristics. Original magnifications 21,000× (survey) and 52,000× (detail) (see Wang2006 - Flavivirus Activation of pDCs and TLR7 Signaling).
Contradictions & Debates
★ Morphology is not identity. No immunolabelling was performed — no immunogold against E protein to confirm the particles are virions, and no marker against an endosomal protein (EEA1, LAMP1) to confirm the compartment. Both assignments rest on ultrastructural appearance: “enveloped particle of the right size and density” and “vacuole with Golgi/smooth-ER membrane characteristics.” That is a reasonable call by an experienced microscopist and it is how the field routinely reports such observations, but it is weaker than a labelled result and the wiki should not upgrade it. The compartment identification in particular is a judgement about membrane morphology, and the source itself describes the vacuoles by their Golgi/ER characteristics rather than naming them as a defined endosomal population.
No quantification. TEM as used here is existence proof, not frequency. Nothing establishes what proportion of cells or of input virions took this route, so the images cannot support any claim about the efficiency of endosomal delivery — which is relevant, since the same paper shows dengue needs ~50× the MOI of influenza for comparable Type I Interferon output.
Related Pages
Plasmacytoid Dendritic Cell, TLR7, Immunohistochemistry, Multi-color Immunofluorescence, Toll-like Receptor Signaling in B Cells