Preparation of Autoradiograms for Electron Microscopy

O'Brien, R. T.; George, L. A.

doi:10.1038/1831461a0

Cited by 46 publications

(6 citation statements)

References 3 publications

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“…However, current approaches lack the ability to distinguish radiotracer uptake in individual living cells. For instance, film autoradiographs can be examined with light [6] or electron microscopy [7] to visualize radioactive decay within individual cells but the method is limited to fixed tissues and low energy radionuclides (e.g. 14C and 3H).…”

Section: Introductionmentioning

confidence: 99%

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

Pratx

Chen²,

Sun³

et al. 2012

PLoS ONE

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Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers.

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Section: Introductionmentioning

confidence: 99%

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

Pratx

Chen²,

Sun³

et al. 2012

PLoS ONE

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“…Using whole bacteria labeled with tritiated thymidine, van Tubergen (4) was able to demonstrate a considerably higher autographic resolution than was shown in the attempts mentioned (3,5) and a somewhat better resolution than could be obtained in radioautography at the light microscope level.…”

mentioning

confidence: 98%

“…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 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.…”

Section: Introductionmentioning

confidence: 99%

Electron Microscopic Autoradiography

Koehler

Mühlethaler

Frey‐Wyssling

1963

The Journal of Cell Biology

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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.

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Preparation of Autoradiograms for Electron Microscopy

Cited by 46 publications

References 3 publications

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

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

Electron Microscopic Autoradiography

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