Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size,Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

Summary A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 x oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r=0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r=0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r=0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30–100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm× 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Phenidone-ascorbic acid development in electronmicroscopic autoradiography

Summary Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible.Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.     

Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides

The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (3H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (3H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.     

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.     

HIGH-RESOLUTION AUTORADIOGAPHY : II. The Problem of Resolution

The resolution obtainable in electron microscopic autoradiographs, using a photographic emulsion consisting of a monolayer of silver bromide crystals, was investigated theoretically and experimentally. The expected distribution of exposed crystals around a point source was calculated from the geometry of the preparation and from the range distribution of the beta particles emitted by tritium. From such a distribution an autoradiographic resolution of the order of 1000 A can be predicted. From the point source distribution, the expected distribution of grains around bacteriophages labeled with tritium was calculated. This distribution was also measured experimentally in electron microscopic autoradiographs of bacteriophages T-2 labeled with thymidine-H³. The two distributions agreed closely. It was also verified, using the nuclear region in thin cross-sections of Bacillus subtilis labeled with thymidine-H³, that resolutions of the same order were obtained for extended sources. It was concluded that an autoradiographic resolution of 1000 A could be achieved with a presently available commercial emulsion, although emulsions with finer grains might be desirable in some circumstances.     

Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides

Summary The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (³H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (³H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.     

Eine Methode zur quantitativen Auswertung von Autoradiogrammen auf der Grundlage der Durchlicht-Mikro-Photometrie

Zusammenfassung Es wird ein Verfahren zur quantitativen Auswertung speziell solcher Autoradiogramme beschrieben, die vor der photographischen Entwicklung vom untersuchten radioaktiven Präparat abgetrennt werden. Die Durchlicht-Mikrophotometrie erweist sich hierbei gegenüber dem in letzter Zeit vornehmlich verwendeten Auflicht-Reflexionsverfahren als besonders vorteilhaft. — Für die von uns bevorzugt verwendete photographische Emulsion Ilford G 5 werden die im Zusammenhang mit der quantitativen Autoradiographie Tritium-haltiger Präparate wichtigen Zusammenhänge zwischen Lichtabsorption, Silberkorndichte, Elektronenemission und Expositionszeit mitgeteilt.

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A method for quantitative evaluation of autoradiographs using transmitted-light microphotometry

Summary A method is described for quantitative evaluation of autoradiographs, which can be separated from the radioactive specimen before photographic development. For this purpose transmitted-light microphotometry is clearly superior to incident-light microphotometry hitherto in use. — The correlation between light absorption, silver grain density, electron emission and exposition time for the photographic emulsion Ilford G 5 is evaluated using Tritium-labelled specimens.

     

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.     

Soluble compound electron microscope (EM) autoradiography: a resolution source to test redistribution of soluble tritiated compounds during processing

The development of a resolution source that can be labeled with either a soluble or insoluble tritiated compound, and of a method for applying a dry, uniform monolayer of emulsion is reported. Influences due to redistribution of the soluble isotope during emulsion coating were measured by comparing the grain density distributions around the resolution source for soluble tritiated proline (3H-PRO) with that obtained for cross-linked tritiated bovine serum albumin (3H-BSA). The grain density distributions resulting from a standard method of emulsion application (partly gelled/loop method) are compared to that obtained from a dry stripping film. It was found that only the dry stripping film gave a grain distribution which was statistically not different for the soluble and insoluble specimens.     

ANALYSIS OF TRITIUM INCORPORATION INTO INDIVIDUAL CELLS BY AUTORADIOGRAPHY OF SQUASH PREPARATIONS

The relation between tritium content of individual cells and grain count obtained in autoradiographs of squashed cells was investigated. The tissues used were root meristems of Tradescantia paludosa and intestinal epithelium of the mouse. The relation between grain count and tritium content is affected by self-absorption which depends on the thickness of the labeled cell. Therefore, squashed preparations were sectioned to determine the uniformity of thickness of nuclei. In a preparation of mouse cells, thicknesses were 1.18 ± 0.35 µ, and in a preparation of Tradescantia cells, 2.97 ± 0.35 µ. The effects of similar and larger variations in thickness upon grain count were studied in material squashed with different pressures; no marked correlation was found. The lack of correlation is explained by the geometric relation between labeled nuclei and the emulsion. By counting grains and directly measuring tritium content in a glass proportional counting tube in the same preparation, the yield of grains per disintegration was measured in Tradescantia cells and found to be 1 grain for 10.9 disintegrations with AR 10 autoradiographic film and 1 grain for 19.3 disintegrations for NTB nuclear track liquid emulsion. Latent image fading may pose a problem with long exposures; the conditions of its occurrence are as yet not well known.     

A comparison of various procedures for fine grain development in electron microscopic radioautography.

Fine grain development for electron microscopic radioautography was investigated with two types of radioactive specimens: sections of tritiated methacrylate, which provide a homogeneously labeled source for quantitative evaluation of the radioautographic reaction, and sections of 125I-labeled thyroid. Radioautographs were prepared with Ilford L4, Sakura NR-H2, Agfa-Gevaert NUC 307 or Kodak NTE emulsions. The radioautographs were developed with one of several "solution physical" development procedures (Agfa-Gevaert, phenidone-ascorbic acid, p-phenylenediamine developers) or with arrested "direct" developments (D-19b, Elon-ascorbic acid developers). By arresting each development at an early stage of the reaction and at progressively longer time intervals, it was possible to examine the sequence of shapes in the growth of developed silver deposits for each emulsion-development combination. Thus, conditions which resulted in the development of small, round, compact silver deposits were defined for each emulsion. These developments were used in conjuction with gold latensification, a treatment which increases the sensitivity of the emulsions and thus compensates for the lowered sensitivity of fine grain development procedures. The location of the silver deposits in relation to the silver bromide crystals from which they derive was investigated. The emulsion gelatin surrounding the crystals was stained whereas the spaces, which remained after the crystals were dissolved in the photographic fixer, appeared transparent. This analysis permitted the selection of development procedures in which the single or multiple round silver deposits originating from a single crystal will remain within or on the boundary of this crystal. By this method, quantitation of radioautographic reactions composed of small, round silver deposits was studied by using the uniformly labeled 3H-methacrylate sections as a standard source of radiation. The conditions under which grain counting is feasible are discussed.     

Double Emulsion Autoradiography for Identifying Tritium-Labelled cells in sections

The sensitivity of tissue autoradiography can be doubled and the number of false negative cells nearly eliminated by interposing thin tissue sections between two layers of photographic emulsion. A mouse was given 50 μc of tritiated thymidine (SA 2,500 c/M) intraperitoneally and killed 1.5 hr later. A portion of the small bowel was removed, fixed and embedded in methacrylate in the usual way. Sections 2 μ thick were cut and allowed to flatten on water at 40° C. Some sections were used to make conventional single emulsion auto-radiographs and other sections were interposed between two layers of emulsion by first coating slides with NTB 3 emulsion, picking up the sections from a water bath at 18° C, drying, soaking 1 min in benzene, drying, and then dipping again in NTB 3 emulsion. They were exposed at 4° C in a low humidity, 100% CO₂ atmosphere for 10 days, developed and covered in the usual way. There was an average of 20.16 ± 1.4 grains per labelled cell in the double emulsion group compared with 10.6 ± 0.9 grains in the single emulsion group. In the double emulsion autoradiographs there were 55.1 ± 1.65 labelled cells per unit area as compared with 39.8 2 2.0 in the single emulsion group.     

An improved technique for rapid autoradiography of cells and tissue sections

Summary We have demonstrated a method for autoradiography of cell and tissue preparations which is both rapid and safe. The method utilizes only the primary scintillator, PPO, placed under the final emulsion to facilitate activation of the silver grains in the emulsion. Exposure of the autoradiographs is complete under the conditions described within 4 h at ambient temperature. The method is sensitive to exposure time and to the concentration of added radioisotope. The exclusion of volatile, toxic chemicals from the preparations allows the experiments to be performed without any health hazard to the investigator.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

Improved detection of in situ hybridization by laser scanning confocal microscopy.

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.     

QUANTITATIVE TRITIUM AUTORADIOGRAPHY OF MAMMALIAN CHROMOSOMES : I. The Basic Method

A technique has been developed that allows repeated autoradiographs to be made of the isotope distribution in the chromosomes of a single cell. A series of 10 separate autoradiographs were made of a Chinese hamster diploid male metaphase cell which had been labeled with tritiated thymidine during the first 15 minutes of its DNA synthesis period in the previous interphase. Each autoradiograph had low grain densities above the chromosomes so that quantitation was feasible. The separate autoradiographs were photographically combined into a single composite in which grain images were converted to lines oriented at right angles to the chromosome axis. The line densities were then measured with a recording microdensitometer to yield graphs reflecting the isotope distribution along each chromosome. The area under each graph was directly proportional to the total number of grains counted above the corresponding chromosome in the 10 separate autoradiographs. The distribution of isotope along the chromosomes was different for each chromosome, and in some cases homologs also differed in their early labeling patterns.     

RESOLUTION IN ELECTRON MICROSCOPE RADIOAUTOGRAPHY

An analysis of grain distributions around a radioactive line source (consisting of polystyrene-³H) showed that the shape of the distribution was independent of the factors that influence resolution, i.e. section and emulsion thickness, silver halide crystal, and developed grain size. These factors did effect the spread of the distribution, however, and thus the distance from the line source within which 50% of the total developed grains fell. We called this distance "half distance" (HD) and determined it for a variety of specimens. When grain distributions were normalized in units of HD, one could plot universal grain distributions for specimens with radioactive sources of various shapes. The use of HD and the universal curves in interpreting radioautograms is discussed.     

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

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 Ca⁴⁵. The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4°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 µ 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 µ) 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.     

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.