Electron Microscopic Radioautography of Thin Sections: The Golgi Zone as a Site of Protein Concentration in Pancreatic Acinar Cells

Abstract

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO4-fixed, methacrylate-cmbedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days;' at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H a injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.With the rejuvenation of descriptive cytology brought about by electron microscopy, there has arisen a need for high resolution cytochemical methods. Radioautography can, under certain conditions, provide such a method. When the low energy beta emitter tritium (1) is used and when the material under study is reasonably thin, a resolution of the order of 1 micron can be expected (2) with conventional methods, at the light microscope level. Theoretically, much higher resolutions could be obtained by using extremely thin sections as well as extremely thin emulsion layers (3, 4). However, the limitations of the light microscope restrict the improvement that can be achieved by this approach, since fairly thick sections are needed and the finer structural details of the cell are not clearly resolved. The first attempts at direct examination of radioautographs of biological materiaI in the electron microscope were performed by Liquier-Milward on tumor cell nuclei labeled with Co ~° (5) and by O'Brien and George on sections of yeas t cells labeled with Po 21o (3), These attempts were'interesting but mainly exploratory in nature, high resolution being precluded to some extent by the high energy particles emitted in both cases.

Protein Synthesis, Storage, and Discharge in the Pancreatic Exocrine Cell

“…It was improved in space by the use of thin sections and thin emulsion films (10) and, in time, by the use of pulse labeling obtained by intravenous, rather than intraperitoneal injection of the label. Under these conditions, we were able to demonstrate a well defined third stage, i.e., the concentration of label within the centrosphere region, prior to the labeling of the zymogen granules (45,46) (Fig. 2).…”

“…It seems likely, therefore, that the condensing vacuoles are distributed among various fractions mainly according to their density, that is to say, according to their content. Since, as was already shown in our earlier work (45,46,9), these vacuoles contain, at certain times after injection, most of the label in the cell and have, therefore, an enormous specific activity, any interpretation based on the results of fractionation studies alone would be erroneous. The present autoradiographic study was, therefore, started in an effort to clarify some of the many problems left unsolved by the studies mentioned.…”

Age changes in the canine pancreas: histomorphological, electron microscopic, and biochemical study

“…The radioactivity appearing later at other sites can be due to migration of the newly synthesized proteins (Warshawsky et , 1963). That newly synthesized proteins move from the endoplasmic reticulum to the Golgi zone has been demonstrated by these techniques (Caro, 1961;Warshawsky et al, 1963;Caro and Palade, 1964;Jamieson and Palade, 1967a, b). Caro (1961) and Caro and Palado (1964), using the guinea pig, reported that incorporated leucine-H^ first appeared in the endoplasmic reticulum of the acinar cells five minutes after injection of the label into the blood stream.…”

“…That newly synthesized proteins move from the endoplasmic reticulum to the Golgi zone has been demonstrated by these techniques (Caro, 1961;Warshawsky et al, 1963;Caro and Palade, 1964;Jamieson and Palade, 1967a, b). Caro (1961) and Caro and Palado (1964), using the guinea pig, reported that incorporated leucine-H^ first appeared in the endoplasmic reticulum of the acinar cells five minutes after injection of the label into the blood stream. Fifteen to twenty minutes after injection the label was concentrated in the Golgi region, lihen the label first appeared it was local ized primarily in the cell regions occupied by the rough surfaced elements of the endoplasmic reticulum.…”

“…How ever, this material often occurs in the form of a dilute solu tion of protein. The fate of these newly synthesized proteins has been the subject of numerous investigations (Sjostrand and Hanzon, 1954c;Palade, 1958b, 1960;Caro, 1961;Warshawsky et a^., 1963;Caro and Palade, 1964).…”