TEM & SEM
Particles adsorbed to a glow-discharged carbon film are embedded in a thin layer of heavy-metal stain, which outlines their shape at about 2 nm resolution. Fast, cheap and the standard check of sample quality.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Negative staining
Particles adsorbed to a glow-discharged carbon film are embedded in a thin layer of heavy-metal stain, which outlines their shape at about 2 nm resolution. Fast, cheap and the standard check of sample quality.
How it differs from the standard method
- Dilute until particles rarely touch; avoid phosphate buffers with uranyl stains.
- Blot to leave stain about as deep as the particles.
- Uranyl acetate or formate for contrast; methylamine tungstate or other non-uranium stains are alternatives.
1Imaging with electrons
Electrons accelerated through tens of kilovolts have wavelengths of a few picometres, so electron microscopes resolve detail a hundred times finer than light can. In transmission electron microscopy (TEM) a broad beam passes through a specimen thin enough for electrons to cross it (sections of 50–100 nm, or particles on a carbon film) and the image is formed by the electrons that are not scattered away.
Dry and fixed
The column is under high vacuum, so specimens must be fixed and either dried, embedded in resin, or frozen. Every step of that preparation can create artefacts that look like structure.
Heavy metals
Negative stain outlines particles in a glass of uranyl acetate; positive stains bind membranes and nucleic acids; colloidal gold marks antibodies as dense black dots of a chosen size.
Surface and charge
Non-conductive samples charge under the beam unless coated with a few nanometres of metal or imaged at low voltage; higher voltages penetrate deeper and blur surface detail.
Beam damage
Electrons break bonds. Minimal-dose imaging and cryo methods protect delicate specimens; negative stain and resin are more forgiving.
2Negative staining
The quickest way to look at purified particles, viruses, filaments or large complexes. A drop of sample is adsorbed to a carbon film made hydrophilic by glow discharge, washed with water, then covered with a heavy-metal salt such as 2% uranyl acetate, blotted and dried.
3Immunogold and correlative imaging
To localise a protein at nanometre resolution, ultrathin sections are labelled with a primary antibody and then protein A or a secondary antibody coupled to colloidal gold of 5–15 nm. Thawed cryosections (Tokuyasu method) of lightly fixed cells preserve antigenicity best; strong glutaraldehyde fixation and resin embedding preserve morphology but mask epitopes.
Key terms
Practise these as flashcards →A heavy-metal salt that dries around particles so they show as light shapes on a dark background.
A brief plasma treatment that makes carbon films hydrophilic so samples and stain spread evenly.
An ultrathin section cut from a sucrose-infused, frozen, lightly fixed sample, labelled after thawing.
Gold nanoparticles of defined size coupled to protein A or antibodies, visible as dense dots in TEM.
Gold particles per µm² of a compartment, compared between compartments and with controls.
Removing liquid CO₂ above its critical point so no liquid–gas interface crosses the specimen and its surface tension cannot collapse it.
Depositing a few nanometres of metal on an SEM specimen to make it conductive.
Correlative light and electron microscopy: imaging the same cell or structure by fluorescence and by EM.
When to use it
- Checking the shape, size and homogeneity of purified particles or complexes quickly: negative-stain TEM.
- Locating a protein relative to membranes and organelles below the light microscope's resolution: immunogold TEM.
- Imaging cell and tissue surfaces, microvilli, cilia or biomaterial interfaces: SEM; finding rare events first by fluorescence: CLEM.
Compared with alternatives
Images particles frozen in vitreous ice without stain for near-atomic structures; negative stain is the quick screen before it.
Reaches tens of nanometres with fluorescence in hydrated, even living, cells, without metal stains.
Localises proteins in whole cells quickly at light-microscope resolution; the usual first step before immunogold.
Measures particle size distributions in solution in minutes, without seeing individual particles.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Particles adsorbed to a glow-discharged carbon film are embedded in a thin layer of heavy-metal stain, which outlines their shape at about 2 nm resolution. Fast, cheap and the standard check of sample quality.
- Dilute until particles rarely touch; avoid phosphate buffers with uranyl stains.
- Blot to leave stain about as deep as the particles.
- Uranyl acetate or formate for contrast; methylamine tungstate or other non-uranium stains are alternatives.