Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.     

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.     

Incorporation of 3H-oleic acid by the proximal convoluted tubule cells of the chick (Gallus domesticus)

Summary Lipid metabolism in the cells of the renal proximal convoluted tubules (PCT) was investigated in healthy fowls and in fowls with the Fatty Liver and Kidney Syndrome (FLKS). The tissue was fixed at 10–25 min intervals after intravenous injection of ³H-oleic acid. The distribution of autoradiographic grains was analysed by the circle method. In normal cells most of the silver grains were associated with the cytoplasmic organelles. Lipid droplets and Golgi elements had the highest specific activity relative to the nuclear activity, which was little above background level. Lysosome-like bodies and mitochondria had lower values. In the cells of the FLKS-affected birds a large proportion of the grains was located over the lipid droplets, which are abundant in this condition. The specific activity of the cytoplasmic organelles was barely 2-fold higher than the nuclear activity. The results suggest that there is a diminished incorporation of esterified fatty acids by the organelles of these cells and that the excess is transferred to the lipid droplets. The identity of low electron density particles observed in the PCT cells of severely affected birds is discussed.     

QUANTITATIVE AUTORADIOGRAPHIC STUDY OF LABELED RNA IN RABBIT OPTIC NERVE AFTER INTRAOCULAR INJECTION OF [3H]URIDINE

The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [³H]uridine. The highest density of silver grains related to [³H]RNA (27–40 grains/100 µm²) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19–20 grains/100 µm²). Axons (4–5 grains/100 µm²) and myelin (2–3 grains/100 µm²) had the lowest grain densities. 74–83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [³H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.     

Immunoelectron-microscopic localization of diffusible birch-pollen antigens in ultrathin sections using the protein-A/gold technique

Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.     

Immunoelectron-microscopic localization of diffusible Birch-pollen antigens in ultrathin sections using the protein-A/gold technique

Summary Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.     

Helical structures in complex plasma II: Collective interaction

The conditions for the formation and stability of quasi-crystal helical structures formed of charged dust grains of equal size in a complex plasma are investigated. A study is made of both the transverse confinement of such structures by means of an external parabolic potential well and the possibility of their self-confinement or confinement by the field of the smaller background grains. It is shown that, in the presence of dissipation, any initial random distribution of dust grains in a cylindrical external potential evolves into an equilibrium helical structure with a constant pitch angle. It is found that, when the external control parameter is smoothly varied, the pitch angle of the helix changes abruptly and the structure of the quasi-crystal undergoes bifurcations, during which the number of interwoven helices changes. The smaller the confinement parameter and the weaker the attractive forces between the grains, the larger the number of bifurcations. In addition, it is found that, because of the attraction (both noncollective and collective) between the dust grains, stable helical structures may exist not only at zero value of the external confinement parameter, but also at a negative one, and that the collective interaction between the grains not only increases the number of bifurcations but also changes their character.     

ELECTRON MICROSCOPE AUTORADIOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS IN THE RABBIT RETINA MÜLLER CELLS

Rabbit retinas were incubated in medium containing 500 µCi of [³H]leucine for 3 min, and transferred to medium without isotope for another 7, 17, 37, 57, and 117 min. Retinal pieces were fixed in paraformaldehyde and osmium tetroxide and embedded in Epon. Thin sections were autoradiographed with Ilford L4 emulsion, and a quantitative study of silver grain distribution per Müller cell portion, and per Müller cell organelle, was carried out. Grain density per unit area was high over the middle cell portion at each incubation interval. Silver grains were numerous over background cytoplasm (which comprised free ribosomes) but their percentage was constant at all times and their relative concentration low. Silver grains were numerous and highly concentrated, at pulse incubation, over the rough endoplasmic reticulum (RER) and then decreased sharply, but this decline coincided with an increase over the Golgi complex, peaking at 20 min. Another peak appeared over the cell periphery at 60 min. These findings suggest the simultaneous synthesis of two types of proteins in Müller cells; structural proteins in background cytoplasm and proteins of secretory type in the RER.     

Ultrastructural distribution of alpha-bungarotoxin binding sites in the suprachiasmatic nucleus of the rat hypothalamus

Summary The distribution of (¹²⁵I) alpha bungarotoxin (-BTX) binding sites in the suprachiasmatic nucleus (SCN) of the adult female rat was examined by electron-microscopic autoradiography. The ultrastructural distribution of silver grains was analysed by line source, direct point count, and 50% probability circle methods. Real grain distribution was significantly different from that of randomly generated hypothetical grains. Line source analysis demonstrated two populations of sources: one associated with membranes, and one inside neuronal structures. Probability circle analysis of shared grains indicated that membrane-bound-radioactive sources were mainly asssociated with axo-dendritic appositions. Only a small proportion of labeled neuronal interfaces exhibited synaptic differentiations in the plane of section. However, the compartment containing synaptic terminals was the most enriched when comparing real to hypothetical grains. Probability circle analysis of exclusive grains demonstrated that sources that were not associated with neuronal plasma membranes were likely to be within nerve cell bodies and dendrites. It is concluded that the majority of specifically labeled -BTX binding sites in the SCN is membrane bound, and may be associated with axodendritic synaptic transmission. The presence of a significant proportion of the label in the soma and dendrites of suprachiasmatic neurons 24 h after ventricular infusion suggests that some of the labeled binding sites (junctional or nonjunctional) may be internalized within these two compartments.     

Localization of the human myelin basic protein gene (MBP) to region 18q22----qter by in situ hybridization

A restriction endonuclease fragment derived from a cloned portion of human genomic DNA corresponding to the myelin basic protein gene has been used to map the position of this gene by in situ hybridization to human metaphase chromosomes. Ten percent of the radioactively labeled sites observed were on chromosome 18. Eighty-four percent of the grains on chromosome 18 were located within the region corresponding to 18q22----qter. This represents a greater than 10-fold increase in labeling at this position over the background grain distribution found along all of the other chromosomes.     

Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

Using serum from human atopic individuals with a sufficiently high titre of IgE and IgG antibodies to birch- or hazel-pollen allergens and antigens, the localization of IgE binding sites in birch- and hazel-pollen grains was determined by pre- and post-embedding electron microscopic immunoautoradiography with 125I-anti-IgE, whereas the IgG binding sites were localized in ultrathin sections of birch-pollen grains by the protein-A/gold technique. Concerning the distribution patterns of both IgE/IgG binding sites within the pollen grains, no difference could be observed in the dormant pollen grain: Labelling was found in the exine part of the pollen wall and throughout the highly condensed cytoplasm except for starch grains and lipid droplets. The intine part and the germination pores were almost completely unlabelled. In pollen grains which had been soaked in a hypotonic buffer for 15 min, however, IgE binding sites were predominantly localized within the intine and the germination pores. The specificity of the labelling reactions and the observed differences in the localization patterns are discussed.     

DETERMINING THE LABELING INDEX IN AUTORADIOGRAPHY

A method is proposed for estimating the labeling index in autoradiographs. It is based on a model for the frequency distribution of grain counts for those cells which have few grains. The method does not require extensive grain counting and provides reasonable estimates even when the background level is high. It is particularly suited for experiments where tumor cells, labeled in vivo, are, autoradiographed after being cultured in vitro.     

Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization

Cultured seminal vesicle epithelial cells exhibited cytoplasmic immunoreactivity following treatment with anti-insulin antisera. In addition, these cultured epithelial cells were found, by in situ hybridization with a radiolabeled insulin complementary deoxyribonucleic acid (cDNA) probe, to contain an insulin or insulin-like messenger ribonucleic acid (mRNA). Autoradiograms of the hybridized cells exhibited heavy labeling over the cytoplasm and minimal distribution of grains over the nuclei and background areas. These observations indicate that cultured mouse seminal vesicle epithelium contains an insulin or insulin-like peptide as well as the mRNA that is required for its synthesis.     

Effect of Drying on Unexposed Autoradiographic Emulsion in Relation to Background

Unexposed blanks prepared from Kodak AR 10 stripping film were dried by 3 methods: (1) fast, open drying with a fan for 25 min, (2) slow drying in a desiccator for 6 hr, and (3) very slow drying in a desiccator for 24 hr. The number of background grains depended on the mode of drying. Fast drying (method 1) gave 0.7 grain per 100 μ², slow drying (method 2) gave 0.33 grain; very slow drying (method 3), only 0.17 grain. The increase of background after fast drying is assumed to be caused by the rapid shrinkage of wet emulsion. This causes an increase in the intraemulsion pressure which, in turn, sensitizes the silver bromide crystals to cause an increase in the number of developable grains.     

不同供水条件对小麦强、弱势籽粒中淀粉粒度分布的影响

以3个淀粉含量不同的冬小麦品种山农12、鲁麦21和济南17为材料,设灌溉和旱作2种栽培处理,对不同水分条件下小麦强、弱势籽粒中淀粉粒的体积、数目和表面积的分布特征进行了研究.结果表明,小麦强、弱势籽粒均含有A(＞9.8 μm)、B(2.0～9.8 μm)、C(＜2.0 μm)3种类型的淀粉粒,但不同类型淀粉粒的分布状况存在明显差异.在强势籽粒中,淀粉粒的体积和表面积分布均表现为三峰分布,而弱势籽粒中淀粉粒的体积和表面积分布则表现为双峰分布.与弱势粒相比较,强势粒中C型淀粉粒(＜2.0 μm)的体积百分比为7.25%～9.31%,表面积百分比为34.88%～41.51%,而弱势粒的体积和表面积百分比分别为5.33%～6.40%和26.31%～33.54%.强、弱势籽粒中＜0.6 μm和0.6～2.0 μm范围内的淀粉粒数目存在明显差异,强势粒为1.86%～6.13%和83.77%～87.77%,而弱势粒为25.72%～37.42%和52.77%～58.48%.与灌溉栽培相比较,旱作栽培条件下籽粒中B、C型淀粉粒体积和表面积百分比显著增加,而A型淀粉粒体积和表面积显著减少;弱势粒中＜0.6 μm的淀粉粒数目显著增加,强势籽粒中淀粉粒的数目无显著变化.与弱势粒相比较,强势粒中的蛋白质含量较高,C型淀粉粒的体积和表面积所占比例较大,而强势粒中的淀粉含量较低,且A、B型淀粉粒比例也较小.与灌溉栽培相比较,旱作栽培条件下强、弱势籽粒中B、C型淀粉粒体积和表面积百分比增加,蛋白质含量也显著增加,淀粉含量降低.表明水分亏缺能提高籽粒中B、C型淀粉粒体积和表面积百分比及蛋白质含量.     

ELECTRON MICROSCOPE AUTORADIOGRAPHY OF ERYTHROID CELLS USING RADIOACTIVE IRON

The "circle analysis" method of Williams (1969) and a new improved method employing hypothetical grains described in the previous paper have been used to analyze the distribution of autoradiographic grains over erythroid bone marrow cells labeled with radioactive iron, ⁵⁵Fe. The resolution obtainable with this isotope was determined by measuring the distribution of grains about a thin line source. This distribution was also used in calculation of the circle size for the Williams's analysis and the distances of hypothetical grains for the new method. The new method provides estimates for the amount of activity in the regions of condensed and extended nuclear chromatin and for the concentration of isotope at the junction between these two areas. The possible significance of activity in this junctional region is discussed.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.     

A NEW METHOD FOR ANALYZING ELECTRON MICROSCOPE AUTORADIOGRAPHS USING HYPOTHETICAL GRAIN DISTRIBUTIONS

A new method for the analysis of electron microscope autoradiographs is described which has two advantages over other methods. This method provides estimates for the activity in different regions of the section and takes into account the actual geometrical shape and position of different regions within the section. Use is made of the measured distribution of grains about a thin line source to construct a hypothetical distribution of autoradiographic grains which is then compared with the actual distribution of grains. Different activities are then ascribed to various regions until a satisfactory agreement with the autoradiographic grains is obtained using the χ² significance. In the next paper, an example of the use of this method is given for the distribution of grains in the nucleus of erythroid bone marrow cells labeled with radioactive iron, ⁵⁵Fe.     

Absolute quantitative autoradiography of low concentrations of [125I]-labeled proteins in arterial tissue

We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of [125I]-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed [125I]-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural [125I]-low-density lipoprotein [( 125I]-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.