Electron microscopy

Question 1

TEM - how and image

Answer 1

forms image from e- transmitted through specimen (bright areas where e- passes through, dark where e- doesn’t) - 2D image

Question 2

SEM - how and image

Answer 2

Forms image from beam of electrons that bounce off specimen - 3D image

Question 3

preparing for TEM

Answer 3

ultrathin sectioning and metal atom staining or gold labeled antibodies, negative staining, shadowing, freeze fracturing and shadowing

Question 4

Ultrathin Sectioning and Metal Atom Staining - prep work

Answer 4

chemically fixed and stabilized, sliced into ultrathin sections with ultramicrotome: no more than 50–100 nm thick, stained with solutions containing lead and uranium

Question 5

Ultrathin Sectioning and Metal Atom Staining - how and image

Answer 5

enhances contrast of specimen because the lead and uranium give still greater electron density to specific parts of the cell. on image: dark areas where metal atoms are  metal atoms are too big, e- can’t pass through them

Question 6

Ultrathin Sectioning and gold labeled antibodies

Answer 6

Antibodies targeted to a membrane protein, antibodies attached to colloidal gold particles. small, dark granules around periphery of cell are the gold-labeled antibody molecules

Question 7

negative staining

Answer 7

intact specimens are simply visualized in relief against a darkly stained electron dense background

Question 8

shadowing

Answer 8

spraying a thin layer of an electron-dense metal such as gold or platinum at an angle across the surface of a biological specimen

Question 9

freeze fracturing and shadowing

Answer 9

specimens are rapidly frozen, placed in a vacuum, and struck with a sharp knife will edge - fracture along lines of natural weakness—the hydrophobic interior of membranes, in most cases

Question 10

Prep for SEM

Answer 10

sputter coating with metal atoms - gold for example