L4 Emulsion Research Articles

Tissue localization of [ 125I]triiodothyronine in the periorbital area of mice: a microautoradiographic study

A significant retention of [ 125I]triiodothyronine ([ 125I]T 3) in the retrobulbar orbital area of mice has been previously shown. The present study was initiated to determine tissue and intracellular localization of the thyroid hormone in the above area which is concerned in human Graves' disease of the thyroid. Male and female Balb C mice were intravenously injected with 0.1 mL of [ 125I]T 3 (0.2 mCi/gmg). At various time intervals (30 s-10 min) the animals were sacrificed, bled and periorbital tissues were isolated under a dissecting microscope. Three series of samples were prepared: (a) frozen samples for cryomicrotome sections, (b) samples fixed in 10% formaldehyde for paraffin embedded tissues and (c) samples fixed in paraformaldehyde (2%), glutaldehyde (2%) and 0.1 M sodium cacodylate for embedding in Epon-Araldite-DDSA. Sections 5 μ m and 400–600 Å thick for light and electron microscopy, respectively, were coated with Ilford L4 emulsion and exposed for 9–21 days. Light microscope autoradiography demonstrated that [ 125I]T 3 injected intravenously is rapidly transported in the cells of fat tissue of the peribulbar orbital area and tissues with glandular or muscular function: the hormone showed a high affinity for the intra- and extraorbital lacrymal gland cells, the cells of the Harder's gland, those of the sebaceous and meibomian glands of the eye-lids, as well as for local muscular structures. Electron microscope autoradiography showed that radioactivity is already localized inside the cells 30 s after the i.v. injection of [ 125I]T 3 and it is distributed throughout the cytoplasm, with a higher concentration in the vesicles of the Harder's gland cells (rich in lipids and porphyrin), in the endoplasmic reticulum and the mitochondria of the lacrymal glands. 10 min after injection, a shifting of the radioactivity towards the nucleus area was observed. In conclusion, after vivo injection, the thyroid hormone rapidly penetrates the cells of fat glandular and muscular tissues in the orbital area. Intracellularly, the affinity of the hormone for the secretory vesicles, rough endoplasmic reticulum, mitochondria and nucleus suggest that T 3 could play a role in secretory and metabolic functions of the tissues in the retrobulbar orbital area.

Efficiency of grain-density autoradiographs with 125I for the light microscope

SUMMARY Efficiencies of grain-density autoradiographs with 125I for the light microscope were determined using Ilford L4 emulsion and Microdol X development. Calibrated layers of 125I-bovine serum albumin and polymer sections of autoradiographic [125I] micro-scales were used as reference sources. Efficiency was determined as a function of the radiation dose, source thickness and thickness of the absorber foils (e.g. non-radioactive material between the source and the emulsion), respectively. By describing the dependencies of these factors quantitatively this study provides a means for planning autoradiographic experiments.

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by “Solution Physical” or D-19b methods

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by “Solution Physical” or D-19b methods

The Electronographic Color-Magnitude Diagram of Hodge 11

A program of electronographic photometry of star clusters in the Magellanic Clouds is being carried out using the McMullan camera on the Danish 1.5-m telescope at La Silla. The observations are being reduced using the Geneva microdensitometer and data reduction programs. Results are shown for the LMC cluster Hodge 11 (=SL 868), based on measurements of two exposures in both yellow and blue-light, each three hours in length, recorded on Ilford L4 emulsion.

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary.

The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse-labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.

Quantitative investigation of scintillator intensification for light and electron microscope radioautography.

Several reports have indicated that scintillators enhance the intensity of radioautographic reactions. If this were the case, the use of scintillators would shorten the exposure time of radioautographs and the production of radioautographs would be accelerated. It was, therefore, decided to examine the effect of scintillators on the intensity of radioautographs in a quantitative manner over uniformly labeled test specimens. Reaction intensities with or without scintillators were compared using Kodak NTB2 emulsion for light microscopy and Ilford L4 and Sakura NR-H2 emulsion for electron microscopy. In addition, the background fog was quantitatively evaluated. No increase in the intensity of the radioautographs was observed under the following conditions: pretreatment of tissue sections with scintillator solutions before emulsion coating and exposure; treatment with scintillator solutions after emulsion coating but before exposure; treatment with scintillator solutions during exposure; contact with a plastic scintillator film throughout exposure. Heightened reaction intensity was obtained only when scintillators were applied during the histological processing of tissues. The impregnation of tissue blocks with PPO (2,5-Diphenyloxazole) and POPOP (1,4-bis-2-(5-phenyloxazolyl)-benzene) during dehydration, infiltration and Epon embedding produced a 20% increase of reaction intensity in light microscope radioautographs using NTB2 emulsion and a 75% increase in electron microscope radioautographs using L4 emulsion.

Resolution of a gold latensification-elon ascorbic acid developer for Ilford L4 emulsion.

The electron-microscopical autoradiographical resolution of a gold latensification-elon ascorbic acid (GEA) developer for Ilford L4 emulsion was determined experimentally, using radioactive line sources of tritiated albumin (Heijnen and Geuze, 1977). For sections with a thickness of 62 nm (SD +/- 11), which were covered with a carbon layer about 5 nm thick and a slightly overlapping monolayer of L4 silver bromide crystals, the measured half-distance (HD) of resolution was 115 nm. This improvement in resolution, the high efficiency of the GEA developer for L4 emulsion (Wisse and Tates, 1968), and the excellent visibility of the cellular structures under the small silver grains, mean that the L4-GEA combination deserves preverence as a method for quantitative electron-microscopical autoradiography.

Ultrastructural localization of γ-aminobutyric acid receptors in the mammalian central nervous system by means of [ 3 H]muscimol binding

This study utilizes tritiated muscimol binding and electron microscope autoradiography (Ilford L4 emulsion and phenidone development) to localize gamma-aminobutyric acid (GABA) receptor sites in the cerebellum of the rat. In the cerebellar cortex, silver grains were associated with somata and dendrites of basket and stellate cells in the molecular layer, with somata and primary and secondary dendritic shafts of Purkinje cells, axons and terminals of basket cells in the pinceau or basket, initial axonal segments and myelinated axons of Purkinje cells, and dendrites of granule and Golgi cells in the granular layer, and with somata and dendritic shafts of large and small cells in the cerebellar nuclei. These data correspond well to the light-microscope-autoradiographic observations in the cerebellum previously reported [Chan-Palay, V. (1978) Proc. Natl. Acad. Sci. 75, 1024-1028]. Label over GABA receptor sites can be localized to the plasma membranes between pre- and postsynaptic elements at synaptic junctions, of which 88.9% of the samples are axodendritic and the remaining 11.1% are axosomatic. GABA receptor sites are also found along axonal membranes of the GABA-containing basket cell within the basket surrounding Purkinje cells, where true axo-axonal synapses are rare. It is speculated that GABA receptors in the basket may suggest a possibility of the basis for synchronization, either self-inhibition or facilitation within the basket formation, or presynaptic suppression of inhibitory action of basket cell on Purkinje cell.

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

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 approximately 15-20%. For a 500-1,000-A biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of approximately 800 +/- 120 A 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.

Variation in the autoradiographic technique: I. Emulsion-developer combinations assessed by photometric measurement of single silver grains.

Five commonly used developers (Acufine, Rodinal, Dektol, D19 and Microdol X) and two popular nuclear gel emulsions (Kodak NTB-2 and Ilford L4) were tested in combinations for adequacy of development characteristics and the distribution of silver particle (grain) size measured by means of a reflectance microphotometer. All developer-emulsion combinations had some range of development time during which maximum development was accomplished with no occurrence of background grains, with the exceptions of the combinations of NTB2-Acufine and NTB2-D19. Ilford L4 emulsion obtained the narrowest grain size distribution with Rodinal and Acufine, followed by Dektol, and D19. The narrowest size distribution for Kodak NTB2 emulsion was achieved with D19 followed by Acufine, Dektol and Rodinal. Microdol-X had undesirable effects on the integrity of the individual grains of both emulsions. The criteria are discussed for selecting the most advantageous emulsion-developer system for the particular mode of evaluation (visual, photometric, television camera) to be applied to the finished autoradiographic specimens.

A Simplified Technique for Electron Microscope Autoradiography

Two techniques are commonly used in applying nuclear track emulsions to specimens for electron microscope autoradiography: the loop method of Caro and van Tubergen (1962) and the flat substrate method of Salpeter and Bachmann (1964). Both of these techniques are applicable with Ilford L4 emulsion, but the high resolution emulsion Eastman Kodak NTE can be used conveniently only with the flat substrate method since it is difficult to obtain a film of NTE emulsion on a loop. Although the loop method has the advantage that it can be applied directly to grid-mounted specimens, it is difficult to obtain an emulsion layer of uniform and appropriate thickness. The flat substrate method now generally preferred involves coating sections mounted on a glass-supported flat collodion film with liquid emulsion, either by dipping or pipetting, exposing and processing the emulsion, and then transferring emulsion, specimen, and film to a grid. This method allows for a uniform application of emulsion to the section, but the final stripping of the complex of collodion film, section and emulsion from the glass slide often leads to specimen damage or loss. For an excellent detailed description of the various steps in specimen preparation, see Kopriwa (1973).

The efficiency of 14C detection in autoradiographs of semithin plastic sections.

Sections of uniformly labeled 14C-methylmethacrylate were used to determine the efficiency of 14C detection by Ilford L4 emulsion layers, 0.22 mu in thickness, in light microscope autoradiographs. When efficiency was expressed as a function of radiation dose (i.e., the number of decays/mu2) there was a strong dependency on radiation dose up to about 6 decays/mu2, after which detection efficiency was fairly constant. For most emulsion batches, the detection efficiency was about 5%, but it ranged from 8 to 3%.

Determination by electron microscope autoradiography of the distribution in plaque of organisms that synthesize intracellular polysaccharide in situ.

The localization and distribution of organisms actively synthesizing intracellular polysaccharide from labelled glucose has been studied in plaque in situ, using electron microscope autoradiography. Freshly extracted teeth were in contact with one of three different glucose concentrations for varying periods of time. After fixation the teeth were prepared for electron microscopy, sectioned, covered with Ilford L4 emulsion and ‘exposed’ for 10 weeks. Electron micrographs revealed that coccal-shaped organisms predominated in the plaques (ages unknown) and that approximately 30% of the total number of organisms were labelled, thus signifying active polysaccharide synthesis. Although fewer in number than the coccal forms, a greater proportion of the filamentous forms were labelled. Regional examination revealed that the proportion of polysaccharide synthesizing organisms was relatively low at the saliva interface (16%) but increased to 52% in the plaque adjacent to the tooth. Plaques subjected to low glucose concentrations failed to exhibit polysaccharide synthesis in cocci beneath the outer microbial layer at the saliva interface. Filamentous forms continued to be labelled much deeper in the plaque. These observations demonstrate that the proportion of organisms actively synthesizing intracellular polysaccharide in mature plaque is much greater adjacent to the tooth than at the saliva interface. It is suggested that acidic environments would be prolonged at the tooth interface, through the catabolism of the polysaccharide. The filamentous forms, although numerically few, probably contribute significantly to this environment during the absence of exogenous sugars. It is further suggested that the low glucose concentration of saliva would be unlikely to provide sufficient energy for microbial metabolism in organisms situated in the deeper regions of the plaque.

The Use of Autoradiography to Determine the Proportion of Bacteria Metabolizing in an Aquatic Habitat

Autoradiographic methods have been developed to determine the proportion of metabolizing bacteria on leaves of Elodea canadensis. Detection with a light microscope of tritium labelling of pure cultures of bacteria was optimal when samples were incubated with 20 μCi of [3H]glucose or [3H]thymidine/ml for 1.75 to 2 h and the bacteria exposed to K2 emulsion for 14 days. Two fluorescent pseudomonads did not form autoradiograms when labelled with [3H]thymidine. For autoradiograms of bacteria from E. canadensis, L4 emulsion was more suitable than K2 emulsion. High-resolution autoradiograms were prepared of a pure culture of a pseudo-monad and of bacteria from moribund leaves of Elodea canadensis. Counts of labelled and unlabelled cells in light-microscopic and high-resolution autoradiograms suggested that valid counts of labelled bacteria could be obtained from light-microscopic autoradiograms. Direct counts of bacteria from E. canadensis leaves of different ages revealed greater increases as leaves matured and died than did indirect methods. Autoradiographic data from young leaves indicated that the percentage of bacteria labelled with [3H]glucose was similar to the percentage of the total population which was estimated by the plate method. On the mature and moribund leaves larger proportions of the populations were labelled with [3H]glucose than were included in the plate counts.

Electron microscope autoradiographic detection of sites of protein synthesis in the rabbit retina Müller cells.

Rabbit retinas were incubated in medium containing 500 microCi of [(3)H]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.

Sensitivity in electron microscope autoradiography. I. The effect of radiation dose.

Sensitivity in electron microscope autoradiography using Ilford L4 emulsion was shown to be affected by radiation dose ( i.e., number of decays in test specimen per unit surface area). The sensitivity tended to be higher with lower doses. This dose dependence was most marked with Microdol X and least with gold latensification-Elon ascorbic acid development. Possible consequences for quantitation in electron microscope autoradiography are discussed.

Ultrastructural localization of zinc in rat exocrine pancreas by autoradiography.

65Zn was infused at a constant rate for 10 days into a rat. Glutaraldehyde fixed, Epon-araldite embedded ultrathin sections of pancreatic tissue were coated with Ilford L4 emulsion and at 211 days exposure were developed. Silver grains were found over the zymogen granules and over the rough endoplasmic reticulum of exocrine cells. Islet tissue was not observed in these studies. The failure of other zinc localization methods to demonstrate zinc in acinar tissue is discussed as are some of the pitfalls of the autoradiographic method and suggestions for future improvement.

Electron Microscopic Radioautography: The Sites of Nuclear RNA Synthesis in Early Embryonic Cells

The relationship between RNA synthesis and ultrastructural organization of chromatin fibrils was examined in the interphase nuclei of the sea urchin embryos (Arbacia punctulata). The embryos at various stages of development were treated for 1 or 3 hours in sea water with 50 μC/ml of 3H-5-uridine. The samples were fixed in OSO4 or glutaraldehyde (GTA) and embedded in Epon or glycol methacrylate (GMA). Radioautographs were made by applying a thin film of Ilford L4 emulsion directly from an expandable wire loop to thin sections mounted on grids. Following exposure of 10 to 20 weeks, they were developed in Microdol-X or physical developer.In the cleavage and early blastula embryos, each interphase nucleus contains diffusely distributed chromatin and shows only very low incorporation of 3H-uridine into RNA (Fig.1). During these periods, many nucleolus-like bodies occur within the nucleus although they are not labeled with the isotope.

Characteristics of three nuclear emulsions for autoradiography at the electron microscope.

On account of their low grain size three commercial emulsions, Gevaert NUC 307, Ilford L4 and Kodak NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers. In order to investigate the sensitivities of the three emulsions, monolayer preparations were irradiated in the electron microscope, using an energy of 5.7 keV corresponding to the mean β-energy of tritium. After exposure the specimens were developed but left unfixed. The sensitivity may then be determined using the percentage of developed grains. For the formation of one latent image the Ilford L4 emulsion must be hit on the average by 1 – 1.4 electrons per AgBr grain; the corresponding figures for Gevaert NUC 307 and Kodak NTE are 2 – 3 and 4 – 5 respectively. The problem of resolution of point and plane sources of radioactivity is discussed. Future advances in microautoradiography will depend on the development of emulsions with lower grain sizes, but such improvement must not be at the expense of sensitivity.

Correlation of grain counts with radioactivity ( 125I and tritium) in autoradiography

The approximate yield per radioactive disintegration was measured for autoragiography with 125I and tritium, using various photographic emulsions. When the isotopes were attached to red blood cells, and autoradiographs made of smears of the cells, the mean grain yields for γ-disintegration from 125I were about 0.44 for L4 and NTB2 emulsions and about 0.21 for AR10 stripping film. In the case of tritium the mean grain yield per β-disintegration was about 0.12 for L4 emulsion. Since the distribution of the isotopes on the surface of red blood cells differs from that likely to be encountered when the label is present within cells in a histological section, grain yields were also examined for these isotopes uniformly distributed in protein films of known thickness and density. Under these circumstances the grain yields from very thin films were very much greater than from thicker films. Over the range of thickness 1 to 5 μ the grain yields from 125I varied very little with the thickness. Using assumptions described in the text, the mean grain yield/disintegration of the isotopes in tissue section cut at 3–5μ, and exposed to L4 emulsion, are estimated to be approximately as follows:—0.23 for 125I and about 0.03 for tritium. The grain yields from constant amounts of isotope incorporated in protein films of varying thickness were also compared with the number of electrons emerging from the films, counted directly in a windowless gas flow counter. With 125I and to a lesser extent with tritium, the number of grains per emergent electron increased with film thickness. It is suggested that this is because the electrons emerging from the thicker films were mainly those in the higher energy ranges given off by each isotope. The implications are discussed for the choice of radioactive isotope in autoradiography of histological sections aimed at detection and detailed localization of small amounts of material.