AUTORADIOGRAPHY. Principle and methodology.pptx

Principle and methodology of autoradiography

1. AUTORADIOGRAPHY

2. What is autoradiography? Radioactive substance  is an entity capable of spontaneous emission of  radiation  in the form of particles or high energy photons resulting from a nuclear reaction. These energy could either be alpha ( α ), beta ( β ) or gamma ( γ ). This substance usually contains unstable atomic nuclei, and are considered to be  radioactive .  Radioactive  decay is a random process that occurs at the level of Individual atoms. Autoradiography is a bio-analytical technique used to visualize the distribution of radioactive labeled substance in a biological sample. It determines the presence and distribution of radioactive isotope. Radioactive material can be localized within a particular tissue, cell and cell organelles.

3. History Niepce de St. Victor was the first to observe autoradiography accidently in 1867 when a blackening was produced on emulsions of silver chloride and iodide by uranium salts. Curies in 1898 had similar studies, and contributed directly to the discovery of radioactivity. After world war II, development of autoradiography as a biological technique was in full swing after the development of photographic emulsions and then stripping film (Rogers, 1979) made of silver halide. At present, biological compound can be labeled with radioactive isotopes opening up many possibilities in the study of living systems

4. P rinciple : Autoradiography is based upon the ability of radioactive substance to expose the photographic film by ionizing it. Sample containing radioactive substance is put in direct contact with a thick layer of a photographic emulsion (thickness of 5-50 mm) having gelatin substances and silver halide crystals (silver bromide, chloride, iodide or fluoride). Alternatively, a piece of photographic film may be used, where section of tissue is allowed to make close contact. After different exposure time, depending on the experiment and the radio active element, the slide or film is developed photographically and examined. All silver halide crystals hit by radiation are reduced to black granules.

5. Silver halide ( AgX ) is ionized by the radiation emitted from radioisotopes, forming Ag+ ions. Ag+ is then reduced and converted to metallic Ag by a developer reagent (usually containing AgNO3), which precipitates within the gelatin emulsion of the X-ray film. The reduction and precipitation are stopped by emerging the film in a fixative solution forming the final image.

6. Factors to Consider Not all radioactive sources are suitable to produce results. It must have sufficient penetrating power to escape from the section and enter the emulsion. Particles with too much energy on the other hand tends to pass through the emulsion without leaving any altered silver grain in their path. A source of suitable half-life must be used. If too great, the exposure will be unduly prolonged . Whereas a substance with half-life only minutes or hours will have decayed before a section is prepared. Tritium (radioactive Hydrogen H 3 ) is one of the substance commonly used for autoradiography. Readily incorporated into wide variety of organic substance (amino acid and carbohydrate) suitable for biological investigation. Others include C 14 and S 35 Better results are obtained with thinner sections and no gap between emulsion and tissue.

7. Reagents. ADHESIVES: Gelatin 1g Chrome Alum 0.1g D/water 200ml STRIPPING FILM: Kodak AR10 or AR50 Liquid Emulsion: Kodak NTB2 ANHYDROUS CALCIUM SULPHATE (dryer) DEVELOPER: Methanol 2.2g Sodium sulphide 72g Hydroquinone 8.8g Sodium bicarbonate 4.8g Potassium bromide 4g Dissolve the salt separately and mix in the order given. Filter before use.

8. STOP BATH Chrome alum 20g Sodium metabisulphide 20g D/water 1L FIXING BATH 30% aqeous sodium thiosulphate 50ml Stop bath 25ml Mix shortly before use. The stock od sodium thiosulphate keeps well.

9. Methodology : In vivo administration Injection or oral administration of radioactive tracer in laboratory. Sample preparation Preparation could either be: whole body or tissue sections Both euthanized animals and section could be cryopreserved. Fix tissue in a simple fixative avoiding mercury and chromium. Process and embed in paraffin wax. Cut thin section 2-5µm and mount them on clean, adhesive smeared dry slides.

10. Take section to water and wash well in distilled water In a dark room, float the film with its emulsion downwards and ensure that it is wider than the slide. Insert the slide beneath the film and draw them obliquely together with the film so that the film overhangs both edges of the slide and its in close contact with the section. Alternatively, make a small quantity of liquid emulsion at 40-45°C and deep the slide in it. Wipe the back of the slide and stand it vertically to drain.

11. Allow the gelatin layer to dry thoroughly in the dark (2-3hrs). Pack the slides in a light proof box in which there is a small quantity anhydrous calcium sulphate. Store the box in refrigerator at 4°C for the necessary length of time. In the dark room remove the slides and develop the emulsion or film at 17-18°C for 2-2.5 minutes. Transfer the slide or film to stop bath for 5min Transfer to fixing bath for 10min Wash well in several changes of distilled water. Counter stain as required. Dehydrate clear and mount the section. NOTE: Step 5,6,8,9 and 10 are done in dark room. Site of radioactive small black granules above section.

12. Fig:2 Showing methodology of autoradiography.

13. Stripping film is uniform in thickness and suitable for precise measurements. While liquid emulsion forms a layer of variable and unknown thickness. On the other hand, liquid emulsion makes closer contact with tissue section. Of the stripping films, A R 10 has thinner emulsion and be preferred for accurate result. A R 50 is more sensitive. Liquid emulsion is expensive and need to be stored in the dark at 4°C. It is wise to arrange the slide with emulsion downward to prevent the slide from dust contamination . Sections must not be packed too close or the radioactivity will contaminate neighbors. When electric incubator or water bath are used in the dark room, the incubator bulbs should be removed and thermostat covered to cancel sparks.

14. Applications Study of different metabolic pathways in tissues specimen and the speed at which it occur eg metabolism of proteins and nucleic acid. To find and investigate the various properties of DNA eg site and time of DNA synthesis in nucleus or mitochondria. To find the location and amount of particular substance within a cell including cell organelle, metabolites etc. Tissue localization of radioactive substance. To find the site and performance of targeted drug.