High-Resolution Autoradiogaphy

Lucien, G.

doi:10.1083/jcb.15.2.189

Cited by 197 publications

(45 citation statements)

References 20 publications

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“…For the purpose of discussing resolution, we consider Kodak NTE and NTE2 to be synonymous.) The Ilford L4-coated autoradiographs were developed for large grains with Gold-EAS (23) or for small grains with paraphenylenediamine (4,5). The Kodak NTE2 autoradiographs were developed with Dektol (Kodak; diluted: one of Dektol to two of water) at 25~ as previously described (19).…”

Section: Discussionmentioning

confidence: 99%

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Salpeter¹,

Fertuck²,

Salpeter³

1977

The Journal of Cell Biology

173

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Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of -15-20%. For a 500-1,000-/~ biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of -800 -120/~ using Ilford L4 emulsion and 500 -70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.On theoretical grounds, 125I is a highly favorable isotope (see Appendix A) for electron microscope (EM) autoradiography. This was early recognized by Kayes et al. (11) in 1962. However, although some calibration studies for both sensitivity and resolution have been performed using this isotope (6, 10), the resolution attainable with this isotope under varying experimental conditions has not yet been systematically assessed. In the present study we have adapted previous calibration procedures used for tritium and ~4C (2,7,20,22) to obtain resolution values for mzsI as a function of section thickness, photographic emulsions, developing procedures, and heayy metal staining as employed in Em autoradiography. The results allowed us to reassess the critical parameters affecting resolution for isotopes of different energy. MATERIALS AND METHODS Resolution SpecimensTwo resolution specimens were used. One was to test resolution in plastic specimens (density 1.1) as a function of section thickness and different emulsion-developer combinations; the second was to test the effect of increasing specimen density by the incorporation of heavy metals such as osmium and uranium, which are used in the fixation and staining of biological material for electron microscopy.RESOLUTION USING PLASTIC TEST SPECI-MENS: The calibration specimen was modeled after that described previously for tritium and 14C (2,20,22) in which a thin film of radioactive polystyrene was sandwiched between a polymerized Epon block and a layer of nonradioactive methacrylate. (The radioactive styrene was thus never exposed to the solvents normally involved in embedding for EM autoradiography, which would

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Section: Discussionmentioning

confidence: 99%

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Salpeter¹,

Fertuck²,

Salpeter³

1977

The Journal of Cell Biology

173

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“…Electron scattering thus greatly improves resolution. Caro (17) and Hill (18) have observed better resolution in autoradiographic studies than can be predicted without consideration of the effect of scatter. Dudley (19) has shown that a diffuse beam of 0.25 Mev electrons produces only about one-third of the grains per unit area that a beam focused perpendicular to the surface of the film does.…”

Section: Theoreticalmentioning

confidence: 99%

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Winegrad

1965

The Journal of General Physiology

126

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Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca 45 . The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4 0°C , during relaxation following K+ depolarization or after prolonged depolarization. From 6 to 39 per cent of K+ contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 /A of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 /j) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.A considerable body of evidence has been accumulated to suggest that calcium plays a critical role in excitation-contraction coupling in muscle. Small amounts of calcium are necessary for maximum contraction and maximum adenosine-triphosphatase activity in muscle models (1, 31) Associated with contraction of intact muscle fibers there is an accelerated exchange of calcium probably involving both membrane and intracellular calcium (2, 3). Hill has shown on theoretical grounds that insufficient time exists between membrane depolarization and muscle activation for a substance to diffuse from the cell surface to the cell center (4). Therefore, the "activator" which directly initiates the contraction must either be transmitted by a process faster than diffusion or must be stored inside the cell at a site near the contractile proteins. Huxley and Taylor have shown that the membrane excitation may be con-455

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“…approach limit the resolution of this technique (17)(18)(19)(20), and will be discussed more fully later. From these two counts the numbers of grains per unit area of organelle or collagen were then determined for each micrograph.…”

Section: Determination Of Availability Of Proline-h ~ After Intraperimentioning

confidence: 99%

Wound Healing and Collagen Formation

Ross

Benditt

1965

The Journal of Cell Biology

284

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The uptake, intracellular transport, and secretion of protein by guinea pig wound fibroblasts was studied by electron microscope radioautography using L-proline-S,4-H a as a tracer. Experiments were performed to determine the curve of concentration of free amino acid in the blood after intraperitoneal administration of the labeled proline. Radioautographs were quantitatively analyzed and the concentration of isotope, in grains per unit area, was determined for the following cellular and extracellular compartments: ergastoplasm, Golgi complex, peripheral cytoplasmic structures, and collagen. The concentration of label, expressed as number of grains per unit area of each subcellular system, reveals the period during which each cellular compartment is maximally labeled, and presents a clearer picture of the passage of the label through each of these compartments. The data demonstrate appearance of the label at maximum concentration in the ergastoplasm 15 minutes after injection, and this compartment remains maximally labeled for 2 hours. In the Golgi complex, concentration is not maximal until 60 minutes after injection of isotope, and appears to decrease before or at about the same rate as that of the ergastoplasm. The present experiment is consistent with previous light microscope radioautographic studies, and no storage phase was found in the fibroblasts. The findings are not simply consistent with a direct precursor-product relationship between the contents of the ergastoplasm and those of the Golgi complex. Morphologic observations of regions in the fibroblast interpretable as possible sites of communication between the ergastoplasm and the extracellular space, together with the kinetic studies, permit the suggestion of an alternate pathway of passage of at least some of the synthesized protein directly from the ergastoplasmic cisternae to the cell exterior.

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High-Resolution Autoradiogaphy

Cited by 197 publications

References 20 publications

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Wound Healing and Collagen Formation

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