EM autoradiography of non‐sectioned biological material

Abstract

Methods of EM autoradiography suitable for the study of non-sectioned biological material deposited on a support film from suspension are described. The methods, which include modified procedures for specimen support and emulsion preparation, may also have advantages for the study of any specimen where physical separation of specimen and emulsion is desirable.In a study of the growth of 1"I-labelled collagen fibrils reconstituted from solution in vitro, these procedures have allowed the production of EM autoradiographs which show a high resolution and are free from stress artefacts and chemography. I N T R O D U C T I O NMost applications of electron microscope autoradiography have been concerned with the study of cell function and its relation to cell structure. In such studies the necessity to preserve in situ relationships makes it imperative that the material should be observed in section. For this reason most of the extensive literature on EM autoradiography describes work using specimens prepared by conventional fixation, embedding and sectioning procedures.But these methods need not always be the most suitable. EM autoradiography can also be a useful tool for studying the separated and purified macromolecular components of a tissue and the way they interact in vitro. A specimen of this nature is less amenable to study in section and more information may be obtainable by other, less conventional, methods of preparation. In this paper we describe such procedures developed for the autoradiographic study of a particular macromolecular system. We show that this approach can yield information on the assembly behaviour of biomolecules and that, in a favourable situation, kinetic parameters of the assembly process may be determined.The modified procedures described here relate principally to the techniques used to support the specimen and to apply the emulsion. For thin-sectioned material there exists a variety of suitable procedures (recent reviews are given by Budd, 1971;Jacob, 1971). Important requirements of any method are (i) reliability and reproducibility, (ii) the production of a layer of emulsion that is flat and of uniform thickness, in close proximity to the specimen and (iii) the avoidance of stress artefacts and chemical interactions between specimen and emulsion. Thin sections of