Autoradiography

Autoradigraphy, In vivo autoradiography, In vitro autoradiography, SPECT, radiolabelled p32 and C14,

1. AUTORADIOGRAPHY Dr. M. THIPPESWAMY

2. INTRODUCTION Autoradiography is the bio-analytical technique used to visualize the distribution of radioactive labeled substance with radioisotope in a biological sample.” • It is a method by which a radioactive material can be localized within a particular tissue, cell, cell organelles or even biomolecules . • It is a very sensitive technique and is being used in a wide variety of biological experiments. • Autoradiography, although used to locate the radioactive substances, it can also be used for quantitative estimation by using densitometer.

3. HISTORY • The first autoradiography was obtained accidently around 1867 when a blackening was produced on emulsions of silver chloride and iodide by uranium salts observed by Niepce de St. Victor. • In 1924 first biological experiment involving autoradiography traced the distribution of polonium in biological specimens. • The development of autoradiography as a biological technique really started to happen after World war II with the development of photographic emulsions and then stripping made of silver halide. • Radioactivity is now no longer the property of a few rare elements of minor biological interest (such as radium, thorium or uranium) as now any biological compound can be labeled with radioactive isotopes opening up many possibilities in the study of living systems

4. PRINCIPLE   • Autoradiography is based upon the ability of radioactive substance to expose the photographic film by ionizing it. • In this technique a 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 ( AgX – silver bromide, chloride, iodide or fluoride – AgBr , AgCl , AgI or AgF , respectively). • This emulsion differs from the standard photographic film in terms of having higher ratio of silver halide to gelatin and small size of grain. • It is then left in dark for several days for proper exposure. • The silver halide crystals are exposed to the radiation which chemically converts silver halide into metallic silver (reduced) giving a dark color band. • The resulting radiography is viewed by electron microscope, preflashed screen, intensifying screen, electrophoresis, digital scanners etc.

5. METHODOLOGY The radioactive sample is covered with the photographic emulsion by several described method. The radioactive part of the sample activates the silver halide crystals near by. This results in reduction of Ag+ ions to Ag atom leaving dark color bands. The slide is then washed away by fixers to get insoluble Ag atom only. The autoradiogram can further be viewed and observed under the microscope.

6. A 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. General principle of autoradiography

7. Autoradiography: Methods Classic autoradiography techniques are performed according to the following general sequential steps : In vivo autoradiography 1. Radioactive labelling of biological sample. Labeling time depends on the type of radioisotope and the radiotracer molecule. Injection or oral administration of radioactive tracer in laboratory animals 2. Sample preparation Cryopreservation of euthanized animals and cryosection – whole-body or tissue sections (20-50 µm thick) for microscopy evaluation. Light or electron microscopy can be used, depending on the aim of the study. Whole-body or tissue sections are mounted into glass slides and embedded in photographic emulsion to generate a latent image. 3. Image development. Here, the incubation time in the developer reagent depends only on the radioisotope used. 4. Arrest of image development by exposing the slide to a fixative reagent.

8. Whole-body autoradiography (WBA) and Quantitative Whole-body autoradiography (QWBA) – A drug distribution in rodents.  A  – intravenous administration of [ 14 C]-ethanol (Gifford, Espaillat , & Gatley , 2008)darker areas correspond to areas of high ethanol concentration.  B  – intravenous injection of [ 3 H]-glucose ( Potchoiba & Nocerini , 2004). Region of high glucose (radioactivity) concentration are shown in green/blue.

9. Rate of DNA replication The rate of DNA replication in a mouse cell growing in vitro was measured by autoradiography as 33 nucleotides per second. The rate of phage T4 DNA elongation in phage-infected E. coli was also measured by autoradiography as 749 nucleotides per second during the period of exponential DNA increase at 37 °C.

10. Detection of protein phosphorylation Phosphorylation means the posttranslational addition of a phosphate group to specific amino acids of proteins, and such modification can lead to a drastic change in the stability or the function of a protein in the cell. Protein phosphorylation can be detected on an autoradiograph, after incubating the protein in vitro with the appropriate kinase and γ-32P-ATP. The radiolabeled phosphate of latter is incorporated into the protein which is isolated via SDS-PAGE and visualized on an autoradiograph of the gel.

11. Detection of sugar movement in plant tissue In plant physiology, autoradiography can be used to determine sugar accumulation in leaf tissue. Sugar accumulation, as it relates to autoradiography, can described the phloem-loading strategy used in a plant. For example, if sugars accumulate in the minor veins of a leaf, it is expected that the leaves have few plasmodesmatal connections which is indicative of apoplastic movement, or an active phloem-loading strategy. Sugars, such as sucrose, fructose, or mannitol , are radiolabeled with [ 14 C], and then absorbed into leaf tissue by simple diffusion.

12. Factors determining the efficiency of Autoradiography Energy of emitter: Higher the energy longer is the track length and so it’s difficult to localize the points in the low density region of the same track. Further very low energy radiation also creates a poorer resolution image on the film. Therefore weak b-emitting isotopes (3H, 14C and 35S) are most suitable because the energy of radiation is in between g and a radiations. Distance and Thickness of sample : If either the sample is very thick or the sample is far away from the emulsion film, resolution will be lost. Grain size and amount of silver halide crystals : The grain size should be smaller so that there is more availability of AgX crystals. Also concentration of gelatin should be less in emulsion as comapred to AgX crystals.

13. 4. Thickness of emulsion: The emulsion thickness affects the efficiency of autoradiography with different emitters. For b-emitters the thickness of the emulsion should be less. 5 . Exposure time : An autoradiogram must be exposed for a sufficiently long time for proper exposure to view pattern of the track length. Vector At5g23940

14.   • To find and investigate the various properties of DNA • 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. • To locate the metabolic activity site in the cell. Applications Of Autoradiography

15. Positron-emission tomography (PET) It is a nuclear medicine functional imaging technique that is used to observe metabolic processes in the body as an aid to the diagnosis of disease. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radioligand , most commonly fluorine-18, which is introduced into the body on a biologically active molecule called a radioactive tracer. Different ligands are used for different imaging purposes, depending on what the radiologist/researcher wants to detect. Three-dimensional images of tracer concentration within the body are then constructed by computer analysis. In modern PET computed tomography scanners, three-dimensional imaging is often accomplished with the aid of a computed tomography X-ray scan performed on the patient during the same session, in the same machine.

16. Positron Isotope Half-life (min) Application 11-carbon ( 11 C) 20.4 Detection of tumor margins and metastasis 13-nitrogen ( 13 N) 9.96 Myocardial perfusion imaging 15-oxygen ( 15 O) 2.07 Hemodynamics and blood flow 18-fluor ( 18 F) 109.7 Tumor and metastasis detection 68-gallium ( 68 Ga) 68.3 Detection of neuroendocrine tumors Common positron emitting radioisotopes used in PET

17. Single-photon emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique using gamma rays and is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required. The technique needs delivery of a gamma-emitting radioisotope (a radionuclide) into the patient, normally through injection into the bloodstream. On occasion, the radioisotope is a simple soluble dissolved ion, such as an isotope of gallium(III). Most of the time, though, a marker radioisotope is attached to a specific ligand to create a radioligand , whose properties bind it to certain types of tissues. SPECT: Single-photon emission computed tomography

18. Radioisotope Half-life Application 67-gallium ( 67 Ga) 78 hours Bone disease  99-technecium ( 99m Tc)  6 hours Bone scanMyocardial perfusion scan Brain scan 111-indium ( 111 In) 2.81 days White cell scan 123-iodine ( 123 I) 13 hours Neuroendocrine or neurological tumor scan 131-iodine ( 131 I) 8.06 days Neuroendocrine or neurological tumor scan Common gamma-ray emitting radioisotopes used in SPECT

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