Electron Microscope Autoradiography of Erythroid Cells Using Radioactive Iron

Incorporation of [nH]threonine and [3H]glucose by the mucous and serous cells of the human bronchial submucosal gland has been studied over 8 h using, for the first time, in vitro pulse labeling and electron microscope autoradiography. In assessing the autoradiographs, two methods were compared, the circle analysis and the recently described hypothetical grain analysis.Preliminary studies showed formaldehyde to be the most suitable fixative. Chemical analysis of tissue revealed that [3H]threonine was incorporated into the polypeptide moiety of the bronchial gland product and that metabolites of [SH]-glucose were incorporated into the carbohydrate.Tritiated threonine was first localized in the endoplasmic reticulum of both mucous and serous cells and later migrated to the Golgi apparatus, while metabolites of [3H]glucose localized first mainly in the Golgi apparatus. From here, both radioactive precursors were next identified in vacuoles and, finally, in secretory granules. The mucous cell incorporated strikingly more of both radioactive precursors than the serous cell. Thus, it seems that oligosaccharides of mucous and serous cell glycoproteins are synthesized mainly in the Golgi apparatus and added there to the polypeptide core which is synthesized in the endoplasmic reticulum.The relationship of the mucous cell to the serous cell is discussed. It seems that under "normal" conditions each cell represents a different line but that injury may transform a serous cell into a mucous cell.The human bronchial submucosal gland includes two important types of secretory cell, the mucous and serous. These cells have different ultrastructural features (33) and have been shown by serial reconstruction to comprise their own individual and discrete regions of the gland (34). Histochemical studies have shown that the mucous cell contains four different types of acidic glycoprotein (25) that may occur either alone or in certain combinations but are not found together in a single cell. In the serous cell, however, both sialo-and sulfomucin always occur together (26). In chronic bronchitis, the pattern of hypertrophy of mucous acini is rather different from that of serous acini 320

Section: Hypothetical Grain Analysismentioning

confidence: 90%

In vitro incorporation of (3H)threonine and (3H)glucose by the mucous and serous cells of the human bronchial submucosal gland. A quantitative electron microscope study.

Meyrick¹,

Reid²

1975

“…This allowed us to obtain a "universal curve" as previously done for the tritium. The tritium universal curve had given a reasonable fit to the data for 14C (22) and also for 55Fe (15), except for a discrepancy in the tail of the distribution (the higher energy isotope 14C having the longer tail). We therefore first tried to fit the tritium curve to the 1251 composite histogram (broken line curve in Fig.…”

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

173

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

“…In both our present paper and Blackett and Party's 1977 paper (4), overlays are constructed using the integrated universal curve for a point source derived from the resolution studies of Salpeter et al (17). Both these studies are also based on the idea for EM autoradiographic analysis initiated in the density distribution method (5,17,20), and elaborated in the hypothetical grain method (3,11,12). In principle, this idea involves hypothesis testing whereby the distributions of observed developed grains in one's autoradiographs are compared with those expected for various defined sources until a "best fit" source is found.…”

Section: Discussionmentioning

confidence: 99%

Resolution of electron microscope autoradiography. IV. Application to analysis of autoradiographs.

Salpeter¹,

McHenry²,

Salpeter³

1978

137

The previous publications of this series described the expected grain distributions around model radioactive structures in EM autoradiographs as a function of the specimen resolution. This family of expected distributions was called the "universal curves". In the present study, experiments on ~4C-labeled specimens refine our information regarding the tails of the universal curves. When the expected grain distributions from 125I-, all-, and ~4C-sources were compared, significant differences were found depending on the energy of the isotope. These differences were primarily in the tails of the distributions, and are therefore important in correcting for cross-scatter when analyzing electron microscope autoradiographs.