Description
This advanced lecture note on Autoradiography provides a detailed exploration of the technique for tertiary students in Biomedical Sciences, Histopathology, and Molecular Pharmacology. Authored by Ephraim Imhotep Zulu, BSc BMS, MSc Path, from the Department of Biomedical Sciences at the University of Zambia's School of Health Sciences, the material defines autoradiography as a specialized biological tool where the specimen itself, having incorporated a radioactive substance, acts as the radiation source to expose a photographic emulsion. The lecture explains the principle based on the ionization of silver halide crystals by radiation, resulting in metallic silver deposits that form an image. It distinguishes between in-vivo autoradiography, where radioligands are administered systemically to living tissue, and in-vitro autoradiography, where slide-mounted sections are incubated under controlled conditions. Key practical applications are outlined, including tracing drug metabolism, mapping receptor locations in tissues, measuring DNA synthesis, and neuronal tracing. The note covers the types of photographic detection systems, such as stripping film and liquid emulsion, and discusses common problems like incorrect localization due to emulsion movement, fogging from light exposure, chemical contamination, or excessive background radiation. This comprehensive guide is essential for diploma and degree students to understand the methodology, applications, and critical limitations of autoradiography in research and diagnostic contexts. Access this lecture to gain expertise in this powerful technique for visualizing and quantifying radioactive labels in biological systems.
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Ephraim Imhotep Zulu, BSc BMS, MSc Path University of Zambia School of Health Sciences Dept. of Biomedical Sciences Cellular Pathology Lecture #20 Advanced Histological Techniques: Autoradiography Lecture Outline • Principle of Autoradiography • Sequential Steps Of Autoradiography • General Methods For Autoradiography • Autoradiography Practical Applications • Limitations of Autoradiography • Types of photographic detection systems • Problems that can occur in autoradiography Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Learning Objectives: At the end of this lecture, the student is expected to: • Know and understand the principle of Autoradiography • Appreciate how biological specimens are prepared for Autoradiography • Compare and contrast: • Radiography vs. Autoradiography • In vivo autoradiography vs. ex vivo autoradiography • Appreciate the practical applications of autoradiography • Know the Limitations of Autoradiography • Appreciate Types of photographic detection systems • Know the common problems that can occur in autoradiography Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Synopsis • Radiography is the visualisation of the pattern of distribution of radiation. • In general, the radiation consists of X-rays, gamma or beta rays, and the recording medium (medium for converting radiation into an image) is a photographic film. • For classical X-rays, the specimen to be examined is placed between the source of radiation and the film, and the absorption and scattering of radiation by the specimen produces its image on the film. Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Radiation source Radiographic film Object Image • In contrast, in Autoradiography, the specimen itself is the source of the radiation, which originates from radioactive material incorporated into it. • The recording medium which makes visible the resultant image is usually, though not always, photographic emulsion. • Therefore, Autoradiography is a specific biological tool used to detect radioactive materials by using X-ray photographic films. • Autoradiography is employed as an important detection tool for the identification of different target receptors in various tissues to provide us with a better understanding of molecular pharmacological pathways. Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY • Autoradiagraphy is based upon the ability of a radiaoactive substance to expose the phtographic film by ionizing it. • In this technique a radioactive substance is put in direct contact with a thick layer of a photographic emulsion (5-50 um thick) having gelatin substances and silver halide crystals. • 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 the dark for several days for proper exposure. • The silver halide crystals are exposed to the radiaton which chemically converts silver halide into metalic silver (reduced) giving a dark colour band. • The resulting radiography is viewed by electron microscope, preflashed screen, intensifying screen, electrophoresis, digital scanners, etc. Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Principle of Autoradiography Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY Autoradiography Method., Sequential Steps of Autoradiography Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY exposure of living cells to a radioactive material. Dissection of samples into sections for coverage with thin film of photographic emulsion incubation of sample in the dark for few days for radioactive decay. Development of photographic emulsion. pre-staining of the entire tissue with osmium. counter staining using Toluidine blue to reveal tissue histology Microscopy to determine the relative position of the silver particles. Generation of records in the form of autoradiographs. Tuesday, July 19, 2022 Ephraim Zulu - HISTOPATHOLOGY The radioautography images of 125I-labeled PPP. a, 5 min b, 18 h and c, 24 h after injection and merged with X-ray images.
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