Autoradiography Efficient for studying dynamic processes in cells and tissues.

1. Labeling Techniques Autoradiography Efficient for studying dynamic processes in cells and tissues. Molecules biosynthesized with radioactive elements behave in the cells exactly as the unlabeled counterparts. The label is added pre-fix and visualization occurs post embedding. Viewed with TEM or light microscopy.

2. History of Radiography • Niepce de St. Victor observes blackening of silver iodide and silver chloride emulsions by uranium nitrate. • 1869 Bequerel observes the same darkening with opaque paper placed between the uranium nitrate and the emulsions.

3. 1904 London puts a frog immersed in Radium water on a photographic plate. 1956 Technique scaled down for use in EM by Liquier-Milward 1980s 14C- and 3H-labeled leucine are used to measure rates of amino acid incorporation into proteins. The use of autoradiography is also proposed for the measurement of ligand binding and drug interaction.

5. Radioactivity - radioisotopes (elements with higher than normal atomic weight) become more stable elemental isotopes by emitting charged particles.

6. If only electron given off - Beta emission Neutrons and protons - Alpha emissions

7. 14C, 3H, 35S, or 125I is detected in tissue by covering sections with photographic emulsion. The sections are kept in a light-tight box where radioactive disintegrations occur to create latent images. At sites of radioactive material, the radioactive emission acts on the silver halide in the emulsion. Subsequent development and fixation turn radiated silver halide into black grains

8. Macroautoradiographic Film 1. A flexible base 200 µm thick polyester or triacetate 2. The photosensitive emulsion 10 to 30 µm thick composed of silver halide grains dispersed within gelatin. 3. A protective supercoat 1-10 µm thick non-photosensitive gelatin)

9. The grains are 1 µm or greater in diameter; large grains facilitate greater sensitivity, small grains enable finer resolution. The grains consist of silver, bromine, and iodine ions arranged in a crystal lattice. Sulfur-containing compounds are often added in order to form specks of silver sulfide, which increase photosensitivity.

11. TEM 1. Sections are placed on slides then overlaid with emulsion Incubation for required time in light tight box 2. Removal of emulsion and sections from slide 3. Grids placed over sections 4. Emulsion scored around grids and picked up 2 1 4 3

12. Commercial emulsion dipping apparatus

15. A. Exposure of silver bromide crystal to beta particle. B. Exposed crystal developed into filament of silver. Non-exposed silver dissolved C. Emulsion or gelatin removed.

16. Most radionuclides used in autoradiography are ß-emitters. Most commonly used ß-emitters are 3H and 14C. Criteria for choosing nuclide include path length, specific activity and half-life:

17. 3H--90% of radiation stays within 1 µm of its point of entry into the emulsion • 14C--90% of radiation stays within 20 µm its point of entry into the emulsion Resolution of label

18. Lysosomes labeled with 125I-Albumin and acid phosphatase rxn product (arrows)

19. Duck reticulocyte incubated with 3H-leucine

20. Endocytosis 125I-albumin taken up in proximal tubules of rat kidney

21. Defects Trailing caused by uranium contamination Carbon film imperfections and lead interference