Manoj Raje and Gyan C Mishra, Institute of Microbial Technology, Chandigrah, and National Centre for Cell Sciences, Pune, India

Autoradiography is a method of localizing radioactive atoms in a specimen. The method briefly comprises of:

1. Labeling of a suitable molecule with a radionuclide, serving as a tracer.

2. Incorporation of the radioactive tracer into the sample under study.

3. Processing of the sample by some suitable means.

4. Exposure by placing the prepared sample in close approximation with a special photographic emulsion so that the products of radioactive decay in the sample may interact with the silver halide crystals of the emulsion to form latent images.

5. Developing the latent images to convert them into silver grains.

6. Fixing the resultant autoradiogram by washing away unreacted silver halide crystals.

The silver grains thus deposited are visualized and their position noted in relation to the underlying sample structure to try and understand the distribution of the label in the sample. Depending upon the nature of the information sought, autoradiography can be carried out at three levels:

1. Gross level: in the case of samples such as entire organs, large tissue slices, leaves, gels, etc., where the resolution required is of the order perceived by the naked eye* (0.2 mm)

2. Microscopic level: cellular and tissue level localization, resolution of around 1-5 |xm required.

3. Ultrastructural level: subcellular detection and localization with a resolution in the submicron range.

Further discussion will be confined to autoradiography of the microscopic and ultrastructural levels only.

For the successful application of the autoradiographic technique to accurately localize and quantify a biological molecule of interest it is necessary to have detailed information of the biochemical, physiological and pharmacological processes involved in its uptake, distribution and clearance. Also important is the need to have knowledge of the efficiency and resolution obtainable by the experimental technique. Efficiency is defined as the number of silver grains formed per radioactive disintegration in the sample. It depends upon:

• Dose, nature and energy of the radioactive emission.

• Thickness of the sample and emulsion.

• Developing parameters.

The resolution obtainable can be considered in three ways:

• Distance of separation between the points of radioactive decay and grain formed.

• Ability to separate two points of radioactive decay.

• Ability to separate the individual grains formed.

Resolution is also modulated by the nature and energy of the radioactive emission and developing parameters; it also depends upon the size of silver halide crystals in the photographic emulsion.

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