The Use of Radioautography and Electron Microscopy for the Localization of Tritium Label in Bacteria

Tubergen, Robert P. van

doi:10.1083/jcb.9.1.219

Cited by 47 publications

(6 citation statements)

References 18 publications

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“…The time of removal of the cells for radioautography is denoted by integral units development time was used to yield very small, easily resolvable grains and low background. The high resolution was necessary because by 7Tr many large DNA units are in one half of a cell (53) and the resulting grains closer together than for fully labeled cells. Reichert phase and bright field optics were used.…”

Section: Figure 16dmentioning

confidence: 99%

Quantitative Radioautographic Studies on Exponentially Growing Cultures of Escherichia coli

Tubergen¹,

Setlow²

1961

Biophysical Journal

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Exponentially growing cultures of E. coli were examined by quantitative radioautographic techniques to determine the distribution of labeled DNA, RNA, protein, and cell wall among the progeny cells of successive generations. It was found that DNA is in large structures, non-randomly distributed in the progeny. About one-half of the cells have four such structures and approximately one-half contain these four structures plus four smaller ones. These structures show remarkable stability. Fewer than 3.5 per cent of the large structures break in one division time. Protein, RNA, and cell wall are all distributed randomly among progeny cells. The number of units of each component that show random segregation must be 200 or more.

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Section: Figure 16dmentioning

confidence: 99%

Quantitative Radioautographic Studies on Exponentially Growing Cultures of Escherichia coli

Tubergen¹,

Setlow²

1961

Biophysical Journal

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“…In another early attempt (3) O'Brien and George mentioned that this technique led to improved resolution and showed some evidence for this. Using bacteria labeled with tritiated thymidine and a fine grain emulsion (Kodak V-1055), van Tubergen (4) was the first to demonstrate clearly a resolution superior to that obtained by conventional techniques. By using thin sections of tissue labeled with tritium and thin layers of fine grain emulsions (Ilford K-5 and L-4), we have obtained autoradiographic resolutions at the subcellular level with little loss in image quality (5,6).…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Autoradiogaphy

Lucien

1962

The Journal of Cell Biology

197

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The resolution obtainable in electron microscopic autoradiographs, using a photographic emulsion consisting of a monolayer of silver bromide crystals, was investigated theoretically and experimentally. The expected distribution of exposed crystals around a point source was calculated from the geometry of the preparation and from the range distribution of the beta particles emitted by tritium. From such a distribution an autoradiographic resolution of the order of 1000 A can be predicted. From the point source distribution, the expected distribution of grains around bacteriophages labeled with tritium was calculated. This distribution was also measured experimentally in electron microscopic autoradiographs of bacteriophages T-2 labeled with thymidine-H 3. The two distributions agreed closely. It was also verified, using the nuclear region in thin cross-sections of Bacillus subtilis labeled with thymidine-H 3, that resolutions of the same order were obtained for extended sources.

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“…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.…”

mentioning

confidence: 99%

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

Lucien

1961

The Journal of Cell Biology

173

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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.

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The Use of Radioautography and Electron Microscopy for the Localization of Tritium Label in Bacteria

Cited by 47 publications

References 18 publications

Quantitative Radioautographic Studies on Exponentially Growing Cultures of Escherichia coli

Quantitative Radioautographic Studies on Exponentially Growing Cultures of Escherichia coli

High-Resolution Autoradiogaphy

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

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