CSF CH. 9B Electron - Microscopy

transmission electron microscopy (TEM)

-uses electrons instead of light to cast images

-MUCH better resolution than traditional light microscopy

-occurs in a vacuum and the specimen must be stained/treated in an extensive process (dead sample)

prep for TEM

-samples fixed with glutaraldehyde and held in place

-Osmium tetroxide used to stain specimens and is electron dense

Immunogold EM

-uses antibodies that are linked to gold particles to localize molecules

-counterstaining with a heavy metal gives the image more contrast (osmium, uranium, gold)

SEM: 3D surface images

-specimen rapidly frozen and coated with a thin metal film

-electron beam shot at specimen and electrons bounce off and give image of the specimen

-resolution - 10 nm

-detailed 3D surface images of specimens

-can use to image cellular sub-components

TEM: 2D with a single image

-multiple TEM images at different angles canbe digitally combineand color coded to create a 3D image

-can see some internal images

Negative staining

a heavy metal salt “stain” is deposuted where there is not surface features

-allows for visulaization of specimens

-put the less electron dense specimen on a background of film with electron dense materials which appear black

Single particle reconstruction

hundreds of 1000s of images of many identical molecules digitally aligned and combined

-avoids problem of specimen damaginge if imaging the same molecule many times

-freeze the cell really quickly so there is no damage to the cell in prep

-digitally combined to make a 3D molecule