Electron Microscopic Autoradiography

Abstract

An improved method has been developed for the preparation of autoradiographs for electron optical study. The refinement lies principally in the routine production of uniformly thin layers of photographic emulsion over the tracer-labeled cell sections. This is accomplished by means of a centrifugal spreading mechanism. Maize root tips grown in the presence of H3-thymidine were examined electron microscopically by this technique and were found to display nuclear labeling with impressive clarity. The new procedure utilized here yields objects in which the finest cell structures are easily resolvable without recourse to gelatin digestion techniques. INTRODU CTIONThe widespread interest in introducing more specificity into fine structure research has fostered several recent attempts to adapt the valuable technique of autoradiography for electron optical use. The pioneering work of Liquier-Milward (4), as well as that of George and Vogt (2) and of O'Brien and George (6), established the feasibility of attempting a union of autoradiography with electron microscopy. These workers also indicated a methodology which has been used with somewhat more refinement in more recent work. These methods basically attempted to use well established, conventional autoradiographic procedures with modifications appropriate to the establish ment of a thin, uniform layer of emulsion over the sectioned or whole cellular material.Van Tubergen (10), working with H3-thymidine-fed E. coli, prepared the photographic emulsion by gently blowing a loopful of sol emulsion over his bacteria-covered grids. Caro (1) found that the production of an emulsion bubble and its deposit on the specimen grid gave the most satisfactory results. Hampton and Quastler (3) utilized the wire loop method of George to form emulsion but added the refinement of enzymatic digestion of the gelatin after development. Przybylski (8) simply spread his emulsion with a pipette or fine brush and also used enzymatic digestion to obtain convincing micrographs of labeling in H3-thymidine-fed Tetrahymena. Pelc et al. (7) used a somewhat complicated procedure of mounting the finished autoradiograph on grids only after all processing was completed, but used a simple dipping method for the establishment of the emulsion film. A novel approach to the production of a photosensitive layer was made by Silk et al.

A Study of the Ultrastructural Localization of Hair Keratin Synthesis Utilizing Electron Microscopic Autoradiography in a Magnetic Field

“…They studied with this method the intranuclear localization of thymidine labeled with tritium atoms which are known to emit beta particles with energies varying from 0 to 18 kev. With this method, they were able to demonstrate a marked increase in number of reacted grains in the nuclei of cells (15), although many other investigators have obtained quite satisfactory electron microscopic autoradiographs showing intranuclear localization of tritiated thymidine without the application of a magnetic field (17,19,(24)(25)(26). However, the radioactive tracer used in the present experiments emits beta particles with energies ranging from 0 to 167 kev, almost ten times greater than those of tritium.…”

“…Recent progress in the application of autoradiographic techniques to electron microscopy has made it possible to relate the fine structure of cells to their metabolic processes. Most of the studies utilizing this technique are concerned with the uhrastructural localization of DNA synthesis because of certain favorable conditions; i.e., tritiated thymidine is specifically incorporated in DNA synthesized by cells prior to division (15,17,19,(24)(25)(26). Materials labeled with tritium, a lowenergy beta emitter, are considered most suitable for obtaining high resolutions with this technique, provided that ultrathin sections of tissue as well as ultrathin layers of photographic emulsion are used.…”