Autoradiography and fluorography

Abstract

Autoradiography is a technique widely used in biology and medicine. It uses a photographic emulsion to visualize molecules that are radioactively labeled. The technique provides a convenient method of detection, giving an accurate representation of the distribution of the label within a sample. The image is often referred to as the autoradiograph or autoradiogram. The image is generated through the radioactive decay of the labeled ligand that is bound to a protein and/or nucleic acid found within a tissue or cell or held on an insert solid support (see Table 26.1). For example, it can be used to: ? Identify a specific protein from a mixture separated by polyacrylamide electrophoresis (1) and held on a support: Western blotting (2). ? Identify nucleic acid fragments separated by gel electrophoresis held on a support: Southern blotting (3), Northern blotting (4). ? Identify the distribution of nucleic acids in a tissue section: in situ hybridization (5,6). ? Identify the cellular location of a radioactive drug product introduced into a metabolic pathway in vivo (7). The first autoradiograph was obtained accidentally in 1867 when Niepce de St Victor observed the blackening of a silver chloride and silver iodide emulsion by uranium nitrate. The first biological experiments were undertaken in 1924 and involved the analysis of the distribution of polonium in biological specimens, although the development of autoradiograph as a biological technique did not start until the mid twentieth century following the development of photographic emulsions using silver halides. The quality and accuracy of the image depends on many factors including the autoradiographic technique, the radioisotope employed, the photographic medium used to record the image and the photographic process itself. Autoradiography can be further divided into microautoradiography and macroautoradiography. The former involves the use of liquid emulsions into which a sample is dipped, following exposure and development of the photographic emulsion silver atoms/grains are visualized under a light microscope. Kodak's NTB-2 and NTB-3, and Hypercoat LM-1 (GE Healthcare) are examples of liquid emulsions currently in use. Emulsions such as Hypercoat EM-1 with small grain sizes are suitable for electron microscope imaging. Macroautoradiography involves the use of x-ray or radiographic films to create permanent images that replicate the distribution of a radioisotope within sample. Many vendors provide suitable autoradiography film including Fuji, GE Healthcare (Hyperfilm), and Kodak Inc (X-Omat and BioMax). The macroautoradiographic procedure and sharpness of the resulting image depends heavily on the isotopes characteristics. The mode of decay, the energy associated with the decay and the half-life are all important properties to consider along with the amount of radioactivity associated with the specimen all affect the autoradiograph. © 2008 Humana Press.