Abstracts of the thirty-third annual meeting of the Scandinavian society of electron microscopy

JOURNALOF ULTRASTRUCTURERESEARCH73, 91-133 (1980)

Abstracts of the Thirty-third Annual Meeting of the Scandinavian Society for Electron Microscopy The annual meeting of the Scandinavian Society for Electron Microscopy, " S C A N D E M - 8 0 , " was held in Aarhus, Denmark, June 9-11, 1980. Given below are titles and abstracts of the biological papers presented there.

Some New Approaches to the Study of Cellular Structures at a Molecular Level. FRITIOF

Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024. S. SJOSTRAND,

For the analysis of the structure of cellular components at a molecular level certain requirements must be satisfied. It must be possible to analyse the material without any staining because staining can introduce spurious staining patterns. This means that the imaging must be based on recording the relative mass density distribution in the specimen. Only the dark-field mode of microscopy can then be applied. For conventional electron microscopy dark-field imaging only a small fraction of the elastically scattered electrons that give the dark-field image are utilized. Therefore, the electron dose must be very high and the specimen is severely damaged. The use of the scanning transmission electron microscope, STEM, of the Crewe type is, therefore, a prerequisite for such analysis. The dark-field image is here obtained by collecting 90% of the elastically scattered electron and the imaging is not distorted by phase contrast. The true mass density distribution is, therefore, recorded. STEM is, furthermore, an analytical instrument which allows determining of the relative differences in average atomic number within the specimen and carrying out of an elemental analysis at a molecular level. The beam damage problem can be handled thanks to the perfect control of the beam dose that is possible with STEM. STEM~ therefore, offers theoretically entirely new possibilities for structural analysis at a molecular level. The present problem is a technical problem of preparing the specimens so that the information obtained is meaningful. The new preparatory procedures developed in my laboratory offer a solution to this problem. These methods in their latest stage of development are described and some examples of their application in connection with the analysis in STEM are shown. It now appears realistic to expect that a new era for the analysis of the structure of cells will evolve in the near future.

Prospects for Electron Energy Loss Analysis in Biology. V. E.

COSSLETT,

Cavendish

Laboratory, Cambridge University, Cambridge, United Kingdom. The technique of recording the spectrum of energy losses suffered by electrons in passing through a specimen is complementary to that of X-ray microprobe analysis. Although nearly 40 years old, it has only recently begun to be exploited for elemental analysis. Its range of applicability and limits of sensitivity are becoming clearer: efficiency of signal collection and discrimination extends down through the lighter elements (in principle to hydrogen). So it is being used, initially on inorganic materials, for analysing in the part of the periodic table that is difficult by X-ray recording (2 <10). It also has some advantage up to calcium or even iron, requiring lower electron exposure (radiation damage) for a given signal strength. Applications in biology are now being explored for cell components tagged with, e.g., fluorine, in pollution studies and wherever light elements may be in flux. 91 0022-5320/80/100091-43502.00/0 Copyright O 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Some Effects of High-Molecular-Weight Cryoprotectants on Fluid Secretion in Calliphora Salivary Glands. TUDOR BARNARD,1 BRIJ GUPTA, AND THEODORE HALL, The Biological

Microprobe Laboratory, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, England. In X-ray microanalysis, the addition of hydrophilic polymers to the bathing saline prior to quench-cooling may inhibit ice-crystal seeding and provides a matrix for extracellular electrolytes. We have examined two hypotheses concerning the mechanism(s) by which fluid secretion is decreased by such mixtures. The rate of fluid secretion by isolated salivary glands of Calliphora was decreased as a linear function of polymer concentration. A × 100 supramaximal dose of 5-hydroxytryptamine was ineffective in stimulating the inhibited secretion rate. Thus, the inhibition is unlikely to depend on binding of 5-HT by the polymer. The K + concentration of the primary secretion is a function of the osmolarity of the bathing fluid. No significant increase of K + concentration in the primary secretion was observed when normal Ringer was replaced by a 25% Dextran-Ringer mixture. Also freezing-point depression and K+-sensitive electrode measurements indicated that ionic activities in the Dextran-Ringer were only slightly altered (2 13%) compared to those in Ringer. Therefore the decreased fluid secretion rate cannot be accounted for in terms of an increased tonicity of the cryoprotectant saline mixture. The order of efficiency of different polymers for slowing the secretion rate corresponded to that for freezing-point depression, suggesting a physical rather than chemical basis for the effect on fluid secretion.

Usage of Polyvinylpyrrolidone as a Cryoprotectant in Cryoultramicrotomy of Root Tip Tissues. KAARINA PIHAKASKI* AND LAHJA SEV~US,t *Laboratory of Electron Micros-

copy, Department of Biology, University of Turku, SF-20500 Turku 50, Finland, and +LKBProdukter Ab, Box 305, S-161 26 Bromma, Sweden. Cryoultramicrotomy has been little used as an electron microscopical preparation technique for plant tissues. This is primarily due to the great difficulty of freezing plant cells rapidly. They have to be protected in order to avoid serious ultrastructural injuries. We introduce here a cryoprotection method for slightly fixed plant material with usage of a high-molecular-weight polymer, polyvinylpyrrolidone, PVP. We infused the glutaraldehyde-fixed root tips of Sinapis alba in graded series of buffered PVP (MW 40 000) solutions between 6 and 35%. The total infusion time varied from 2 to 3.5 hr. Solid nitrogen served as the freezing medium. The cryosections were cut using glass knives with LKB 8800 Ultrotome III equipped with an LKB CryoKit. The best cutting results were attained at temperatures of -100 to -90°C for the specimen and -90 to -80°C for the knife. We were able to obtain transversely cut thin sections ca. 0.3 mm in diameter from the root cap and meristem cells in various degrees of vacuolization. The cytoplasm of root tip cells had a " s m o o t h " fine-grained appearance when the sections were stained negatively with uranyl oxalate. In the highly vacuolated root cap cells plasmolysis and shrinkage could not be avoided with long infusion times but in the material infused for 2 hr plasmolysis was not generally observed. The organelles closely correlated with those observed by conventional thin-section technique. The advantages of using PVP compared with sucrose infusion are more even distribution of the negative stain, better sectioning, and lower osmotic activity. 1 Present address: Wenner-GrenInstitute, Norrtullsgatan 16, S-113 45 Stockholm, Sweden.

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Ultrastructural Localization of Metals in the CNS by Physical Development: Gold, Mercury, and Water-Insoluble Metal Sulfides. G. DANSCHER, Institute of Anatomy B, Uni-

versity of Aarhus, 8000 Aarhus C, Denmark. Physical developers are photographic developers that, in addition to reducing molecules, contain silver ions. To prevent a direct reduction of silver in the solution, a protecting colloid is added. Physical developers are used in photography to develop latent pictures. Submicroscopic traces of silver in the photographic plate act as nuclei for the reduction of silver ions to molecular silver, which remains where it is formed. Other metals and metal sulfides can act as nuclei for the reduction of silver, leading to a visualization of their cellular localization. The distribution of metals or metal sulfides can be studied in the electron microscope when the tissue is appropriately treated prior to development. The mercury in tissue from animals treated with mercury compounds appears as a well-localized precipitate of silver after being subjected to physical development. In the same way, gold can be visualized in tissue taken from a gold-treated organism when Epon sections are exposed to uv light before development. Energy dispersive X-ray microanalysis (EDAX) demonstrates identical localization of gold and silver, but it has not been possible to verify the same coincidence for mercury and silver, probably because of the high volatility of mercury. In order to demonstrate metals that cannot be directly visualized in the same manner as gold and mercury, either because they cannot, as gold, be reduced to nuclei-forming molecular metal, or do not, as mercury, exist in the tissue as sulfides or polysulfide complexes, it is necessary to add sulfide ions during the fixation. In this way, available metals are transformed to metal sulfides and can be developed. To exclude false reactions, it is necessary to keep a relatively low concentration of sulfide ions in the solution used for perfusion. It is also important to avoid oxidation of the created metal sulfides and to omit formaldehyde in the fixative. Procedures for ultrastructural localization of gold, mercury, and water-insoluble metal sulfides by physical development will be presented.

Rapid Freezing of Freeze-Etch Specimens. A. ELGSAETER, T. ESPEVIK, AND G. KOPSTAD, Institute of Biophysics, University of Trondheim, N-7034 Trondheim-NTH, Nor-

way. The importance of a high cooling rate when freezing specimens for freeze-etching has long been recognized. The two basic methods for achieving rapid specimen freezing are: (1) dropping of the specimen onto a metal surface at low temperature, (2) bringing the specimen instantaneously into thermal contact with a liquid at low temperature and subsequently maintaining a high relative velocity between the liquid and the specimen. Method 1 making use of high-purity copper at liquid He temperature has generally been considered the ultimate method for specimen rapid freezing and has over the last couple of years received strong renewed interest. However, because of the high price and limited availability of liquid He the method is not well suited for becoming a routine method in most labs. We have conducted a theoretical and experimental comparative study of the cooling rates of the liquid He/copper-block method and the much more economical liquid propane jet rapid freezing method. Our theoretical analysis indicates that for distances exceeding 2-3 /xm from the specimen surface the two methods give virtually identical cooling rates. This is supported by our experimental studies which further show that for aqueous specimens the liquid propane jet method by far gives the most reproducible results. Freeze-fractured erythrocyte membranes frozen using liquid propane jet rapid

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freezer reveal structures not seen after freezing using the liquid He/copper-block device or the standard Freon freezing procedure.

Rotary Replication of Rapidly Frozen Cell Junctions. B. VAN DEURS, Institute of Medical Anatomy, The Panum Institute, DK-2200 Copenhagen N, Denmark. This study presents information about the "native" structure of tight and gap junctions as it appears in freeze-fractured, rotary replicated hepatocyte membranes which have not been chemically stabilized and cryoprotected, or isolated, or in other ways manipulated except for rapid freezing in a nitrogen slush. This freezing technique indeed causes ice crystal formation and severe distortion of the tissue, but apparently well-frozen and undamaged membrane areas occur frequently, especially in the periphery of the replicas. Rotary replication was used to improve the level of structural information in the replicas. To test the advantages of this technique, also staphylolysin (kindly provided by Dr. S. Bhakdi, Institute of Medical Microbiology, Giessen, West Germany), a cylindrical molecule resembling the gap junction connexons, was rotary shadowed and, for comparison, unidirectionally shadowed or negatively stained. It is shown that rotary replication is a reliable technique that reveals structural details and radial symmetry to a much higher extent than unidirectional shadowing. In the rapidly frozen, rotary replicated liver membrane, tight junctions appear very distinctly as a system of aligned particles on the E face (B. van Deurs and J. H. Luft, 1979, J. Ultrastruct. Res. 68, 160-172). Gap junctions appear on P faces as more or less densely packed particles exhibiting a remarkable variation in size and shape not visible in unidirectionally shadowed preparations. Mostly no obvious order can be established on the P faces. In contrast, the regions of E-face pits adjacent to the above mentioned P-face areas are typically arranged in a very regular hexagonal pattern with a lattice constant of 9 nm. These observations show that the gap junction connexons are not aggregated and hexagonally packed because of glntaraldehyde fixation, and also, that a very pronounced plastic deformation takes place during fracturing, completely masking the native regularity on P faces. However, to what extent the hexagonal array reflects the in vivo organization of the connexons is uncertain.

Perfusion Fixation of Human Kidneys for Ultrastructural Analysis. JENS CHRISTIAN MOLLER,* ELISABETH SKRIVER,t T. STEENOLSEN,* AND ARVID B. MAUNSBACH,t tDepartment of Cell Biology, Institute of Anatomy, University of Aarhus, and *University Institute of Pathology, Kommunehospitalet, Aarhus, Denmark. Ultrastructural studies of human kidney tissue have almost exclusively been based on immersion-fixed needle biopsies. This approach is usually acceptable for glomeruli, but results in a less satisfactory ultrastructural preservation of tubules and interstitial tissue and provides only limited amounts of tissue for analysis. We have therefore developed a procedure for vascular perfusion-fixation of surgically removed human kidneys to obtain well-fixed normal or pathologically changed tissue for electron microscopic investigations. Nephrectomy specimens were perfused through the renal artery as soon as possible (3-5 rain) after removal. The perfusions were preceded by a brief rinse with a Tyrode solution and carried out under pressure control with 2% glutaraldehyde in 0.1 M cacodylate buffer containing 2% dextran (MW 40 000). Lissamine green was added to the fixative to indicate uniformity of perfusion. Satisfactory ultrastructural preservation was observed in glomeruli, tubules, and interstitial tissue in the entire width of the cortex and

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allowed both qualitative and quantitative analysis of pathological changes such as tubular atrophy, thickening of basement membranes, and altered tubulocapillary relations. As compared to perfusion-fixed kidneys of experimental animals the brush border region of proximal tubule cells was less regular, presumably due to the interruption of blood flow prior to perfusion. The lateral intercellular spaces were often dilated, particularly in tubules from those cases (tumor kidneys) where the renal vein had been ligated early during the operation. The present observations demonstrate that human kidneys, which have been surgically removed and subsequently perfusion fixed, are suitable for ultrastructural analysis.

SEM and TEM of the Normal Rabbit Aortic Endothelium after Controlled Perfusion Fixation. C. GARBARSCH, J. T~NUM-JENSEN, AND B. VAN DEURS, Institute of Medical

Anatomy, Departments A and C, University of Copenhagen, The Panum Institute, DK2200 Copenhagen N, Denmark. To reveal the fine structure of the thoracic aortic endothelium under normal circulatory conditions we developed a method by which fixation was initiated while the aortic circulation was intact and undisturbed. Constant-pressure monitoring ensured that the luminal pressure was kept within 100 _+ 5 mm Hg. We found it an indispensable condition that the pressure be kept within such narrow limits from the moment of contact with fixative until inelasticity of the vessel wall is achieved. Pressure fluctuations larger than this will disrupt the normal surface structure. As fixative we employed glutaraldehyde at decreasing concentrations (5-1.5%) contained in 0.11 M phosphate buffer, pH 7.3, with Dextran T-70 added (1.6-2.35%) to achieve constant viscosity of the fixative. Aortic segments were postfixed in OsO4 and processed for TEM and SEM by standard methods. The study was confined to the ventral wall of the vessel as the surroundings of the intercostal arteries are never uniform. The endothelial cells are elongate and form a regular pattern of extensive overlay so that the single cell typically has its upstream end shielded by adjacent cells while its downstream end is luminally exposed as a rounded tongue. The thin marginal flaps of two cells which cover their common neighbour may be retracted for a short stretch, whereby an area, equivalent to the stomatas observed in silver stainings, is formed. The bottom of the area is the surface of an underlying endothelial cell. Also, underlying cells may protrude as fiat-ironed mushrooms between neighbouring cells. Such phenomena form locally complex patterns of triple overlay. A faint longitudinal striation of the endothelial cells often observed in SEM is due to numerous slender bundles of intracellular actin-like filaments as revealed by TEM. We always find the luminal surface of the endothelium to be uniformly fiat, except for a slight nuclear bulging and the faintly elevated edges of the marginal flaps. The extensive overlay and the marginal flaps presumably serve to provide sufficient "buffer" area for the expansion of the vessel during the pulse waves, thus allowing the vessel to adapt itself to the rapid variations in the blood pressure.

The (S)TEM Attachment--Future Developments in Biological Applications? B. V. JoHANSEN, National Institute of Public Health, Oslo, Norway. Significant differences in the use of electron optical elements prevent the scanning attachment transmission electron microscope, (S)TEM, to reach a resolution near that of the dedicated Crewe-type STEM instrument. In biology the (S)TEM equipment has had its major impact in morphological and elemental analysis of thicker specimens. There

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are, however, trends in the design and development of commercial (S)TEM microscopes which seem to have the potential of giving morphological information down to 1.0 nm. This presentation expresses the author's views on the development of the (S)TEM applied to thinner specimens of potentially higher resolution. The requirement of a high-brightness electron source is imperative in a high-resolution S T E M instrument. This is obtained with a field emission gun in a dedicated STEM. The equipment is rather expensive and commercial attachments seem to have variable performance. The development of lanthanum hexaboride emitters (LaB~) (see Johansen, these proceedings) with brightness values of the order of - 1 0 '° A m -2 St -1 is capable of - 1 - n m resolution with a reasonable signal-tonoise ratio. Improvements in the vacuum system may be necessary, but as a side effect the general contamination situation will benefit from such modifications. The specimeninduced contamination can be reduced by installing a uv irradiator in the specimen chamber. Radiation damage in biological specimens will be further reduced with second-generation low-dose exposure units. Application of signal processing equipment similar to that used in dedicated instruments will be seen as standard in coming S T E M attachments. The annular dark-field detector, solid state or photomultiplier, will be further developed and applied to the (S)TEM. A multizone annular detector, in combination with arithmetic signal processing circuits and free lens control of the post specimen optics, will allow element detection/discrimination in labelled cell organelles and stained nucleic acid specimens. Slow scan/real time image display converters will become less expensive and be a c o m m o n aid in operating the (S)TEM instruments optimally. The high collection efficiency of the annular detectors allows aldehyde-fixed material to be investigated without adding heavy atom staining agents. In combination with electron energy loss spectroscopy (EELS) light elements will be detectable with higher yield than with existing X-ray methods.

Experience

with a Single-Crystal

LaB6 Emitter in a Transmission

Electron Microscope.

B. V. JOHANSEN, National Institute of Public Health, Oslo, Norway. Lanthanum hexaboride (LaB~) emitter has been used in the scanning electron microscope for several years and offers usually increased brightness and expanded lifetime over thermionic electron guns. In order to test the applicability of a LaB~ gun on a specialpurpose 100-keV transmission electron microscope a single-crystal emitter was purchased from Kimball Physics Inc. The radiation damage imposed on biological specimens by the high-intensity beam was neglected here. The vacuum system was operated at a pressure <6 × 10 7 Torr using LN2 in the reservoir above the diffusion pump. No modifications were made in the cathode heating circuit. A Johansen-type grid cap (B. V. Johansen, 1973, ~licron 4, 121-135) was modified to accommodate the relatively bulky LaB~ tip. The conical part of the grid was made separable from the base by a threaded screw mechanism. There was a twofold reason for this: (a) accurate emitter-to-grid distance could be set and (b) the grid aperture could be easily removed for cleaning. The LaB~ tip was set 0.5 mm behind the front of the 1.2-mm%b aperture of the grid. The filament current was turned to 50% of total heating for 30 min prior to use. Standard cathode saturation criteria were used for proper filament setting. The following features were observed with the LaB6 gun: - - The current density at the specimen level was seven to eight times higher than that --

of a hairpin filament for same beam current and illumination setting. A brightness of - 2 . 5 × 101° A m -~ St -1 was obtained with a 30-tzA beam current.

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- - At full beam the phase contrast transfer characteristics of the objective lens did not

deteriorate to the degree as expected from Boersch effects. - - Beam instabilities and reduced brightness were observed after - 3 0 hr of use. A

combined effect of contaminants in the grid aperture and an off-center emitter tip seem to be responsible for this. With the removable grid cap it was easy to clean the aperture and to center the tip within the holder after replacement so that optimum brightness conditions could be restored. In conclusion, if optimum operating conditions of a 100-keV LaB 6 emitter should be obtained over longer periods (>50 hr) it seems necessary to change the conditions in the gun chamber by one or several of the following improvements: (a) change to ether-based oil in the diffusion pump, (b) add an ion getter pump to the anode chamber, and (c) install a cryo shield in the anode chamber.

F i l m T h i c k n e s s M e a s u r e m e n t s w i t h a S i m p l e V i b r a t i n g Q u a r t z M o n i t o r . B . V . JOHANSEN, ELLEN NAMORK, K. OYGARDEN, AND T. HOLTET,* National Institute of Public

Health and *Marconi Norsk, Oslo, Norway. Several preparation procedures in transmission (TEM) and scanning (SEM) electron microscopy require thickness monitoring of evaporated or sputtered films. The vibrating quartz film thickness monitors have proved to be efficient and reliable. However, many of these instruments are highly automated and hence expensive ($4000-6000). The present paper describes the theoretical background and design of a simple and inexpensive ($300500) model which has the required accuracy and is easy to operate. The principle of any vibrating quartz thickness monitor is that the mass (m~) of a material deposited onto a crystal changes the oscillator frequency (Af) according to

Af = -Cj.F lrn~

(1)

where C~ is a mass sensitivity constant and the negative sign indicates a reduction in frequency. Since rn, = Fpd, Eq. (1) becomes

Af

=

-Cyp~d,

(2)

where p~ is the density of the coating material and d the film thickness. Cs depends on the oscillator frequency (fo) of the crystal, Cf = foe(pkN) -1,

(3)

where Pk is the quartz density (2.65 x 103 kg -a) and N is a cut factor (1.67 kHz m) of the crystal. Equation (2) is therefore generalized to accommodate any oscillator frequency and quartz crystal. For a 6-MHz crystal the deposited thickness d (nm) is given by d =-Af(8.1p~) 1 (Af in Hz; p~ in g c m 3).

(4)

The present thickness monitor is designed with only one oscillator and a frequency counter. A digital frequency meter counting in the range 0-80 MHz (resolution 1 Hz) is used. Low-cost quartz crystals which fit oscillators within this frequency range can be obtained from several manufacturers, e.g., SEI, Ltd., U.K.: QC327; or Marconi Ltd., U.K.: QO1670. These crystals are mounted on 2-pin metal holders which can be obtained with loose metal screening cans. A hole, corresponding in diameter and location to the gold-plated electrode area on the crystal, is drilled on the "up-side" of the screening can.

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The crystal holder fits into a special socket into which the other oscillator c o m p o n e n t s are mounted. The socket assembly is e m b e d d e d in E M - E p o n . The crystal and oscillator assembly m e a s u r e s 20 (w) × 25 (1) × 10 (t) m m 3. A v a c u u m feed-through for two wires is required since the frequency counter is located outside the bell jar.

A Cool Diode Sputtering Unit for Heat-Sensitive Specimens. B. V. JOHANSEN AND ELLEN NAMORK, National Institute of Public Healtk, Postuttak, Oslo 1, Norway. In heat-sensitive materials the morphology often deteriorates during diode sputter coating. The literature is not consistent with regard to the Specimen t e m p e r a t u r e at the end of the sputtering process. It seems to vary b e t w e e n 40 and 200°C. We report here on specimen t e m p e r a t u r e m e a s u r e m e n t s carried out in (a) a conventional diode sputterer where the cathode is positioned 3 cm a b o v e the anode and (b) a new electrode configuration. In the latter setup the aluminum anode is placed concentrically at the same level (height) around the cathode (Au/Pd, 50-ram (~). The electrodes are spaced by a 3-mmwide Teflon insulator ring. The total diameter of the concentric diode a s s e m b l y is 115 ram. In order to monitor the t e m p e r a t u r e a specially made N i / C r - N i t h e r m o c o u p l e was prepared. The wires are 0.12-mm 4~ and to the tip an electron microscope grid (3-mm (~) is welded. Such a thin foil target simulates the surface layer of the specimen. The diode (plasma) current at the specimen level was measured with a F a r a d a y cup. A vibrating quartz monitors the thickness of the sputtered coatings (see Johansen et al., in these proceedings). In all experiments the anodes (specimens) were kept at r o o m t e m p e r a t u r e (-22°C). The standard sputter coater was operated at - 7 5 - r e T o r t pressure, with a voltage of - 9 0 0 V and a p o w e r supply current of - 4 5 mA. The current density at the specimen level was - 0 . 3 5 × 10 3 A cm -2. With the monitors and specimens located close to the center of the anode a t e m p e r a t u r e equilibrium of - 7 0 - 8 0 ° C was reached after 1 min. With the specimens located on the anode just outside the geometric shadow of the Au/Pd target the specimen t e m p e r a t u r e increased to - 4 5 ° C after 1 min. The most dramatic improvement was o b s e r v e d with concentrically oriented electrodes. The operating p a r a m e t e r was - 6 0 - m T o r r pressure, a voltage of 1200 V, and a p o w e r supply current of - 1 5 mA. The current density at the specimen was only - 6 × 10 -~ A cm -2. The t e m p e r a t u r e rose and stabilized at - 3 2 ° C after 1 rain. With the new electrode configuration it is easy to o b s e r v e all the specimens during the coating process which for certain purposes are convenient (see also J o h a n s e n and N a m o r k , in these proceedings). Methyl stearate crystals (mp 39.1°C) were well p r e s e r v e d when sputtered with the new electrode configuration. This was not the case with the standard sputter g e o m e t r y and similar diode current (15 mA).

A "Clean" Sputtering Unit Which Allows in Situ, Direct Drying of Biological SEM Specimens. B. V. JOHANSEN AND ELLEN NAMORK, National Institute of Public Health, Postuttak, Oslo 1, Norway. Occasionally, decoration artefacts can be o b s e r v e d on sputter-coated S E M specimens. Many of these artefacts are probably due to residual h y d r o c a r b o n s and/or water vapour. In order to omit the h y d r o c a r b o n a c e o u s contaminants we have attached an oil-free vacu u m s y s t e m to our sputter coating unit. This consists of an oil-free, reversed c o m p r e s s o r which brings " f o r e v a c u u m " down to - 1 0 0 Torr, followed by a liquid nitrogen-cooled sorption p u m p (Ultek, P e r k i n - E l m e r ) . The end v a c u u m of the latter p u m p is 5-10 mTorr. In our efforts to eliminate the influence of adsorbed water v a p o u r we are suggesting direct drying (DD) of the specimens from F r e o n 113 inside the evacuated bell jar. The DD

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method was originally reported by Liepins and deHarven (A. Liepins and E. deHarven, 1978, Scanning Electron Microsc. 2, 37-43) using a separate desiccator with a pressure - 2 × 10-2 Tort. By doing as reported here, we have a less time-consuming preparation procedure combined with "lowered risks" for resorption of water either during storage or transfer to the coating unit. The dehydration and infiltration of the specimens in Freon 113 is carried out according to the Liepins-deHarven method. At the final stage the specimens are transferred to small aluminum crucibles in which they are totally immersed in Freon 113. The pressure in the bell jar is reduced carefully and the drying process is finished within 3-5 min. The bell jar is then flushed four times with dry argon and pumped down to - 1 0 mTorr each time. The specimens are then coated with Au/Pd using a concentric diode sputter coater (see Johansen and Namork, these proceedings). HeLa cells mounted on glass coverslips are processed according to this procedure. Relative to critical-point-dried specimens it is not possible to distinguish between these cells and those processed by the in situ direct drying method.

Surface Conditioning of Specimen Supports for Biological Electron Microscopy. ELLEN NAMORK AND B. V. JOHANSEN, National Institute of Public Health, Postuttak, Oslo I, Norway. In biological electron microscopy of particulate materials, uneven spreading of the specimen on the carbon support often represents a problem. Glow discharging filmed grids prior to specimen preparation is an approach which is frequently recommended. This method, however, does not always give consistent results. We have tried to analyse the effect of different locations of the coated grids within the glow discharge applying various specimens after the treatment. The glow discharge apparatus consists of two 80ram-q5 aluminum electrodes spaced 40 mm apart. They are placed within a cryosorption pumped vacuum unit with the anode connected to ground and the cathode to the negative terminal of a dc power supply. The grid holder consists of eight small spring loaded clips mounted in an electrically isolated PVC stub. The clips are sprayed with Teflon solution. After roughing the bell jar to 100 mTorr the discharge takes place for 15 sec at 200 mTorr with a current/voltage of 4 mA/375 V. Carbon-coated grids positioned in Crookes dark space are assumed to get a net negative surface charge by short treatments. Ferritin molecules diluted in NaCI, pH 7, will be shielded by Na + ions and consequently this specimen spreads evenly on the negatively charged film. When negative staining is applied, good results are obtained with UO22+ since its net charge is positive. If the ferritin solution is brought below its isoelectric point, it will be shielded by C1- ions which results in clustering of the ferritin particles and uneven paths of stain. When the grids are placed on a Teflon disk below the anode (or away from the electrodes) the observed, well-spread DNA, which acts as a polyanion, indicates a net positive charge on the film. The results are consistent, but the mechanisms involved are not fully understood. Brittle carbon films, often reported after glow discharge, were not experienced. This could be related to the short treatment (15 sec) and the low voltage applied.

Fixation and Mordanting Effect of Tannic Acid on Plant Cell Ultrastructure. PETER

OLESEN, Institute of Plant Anatomy and Cytology, University of Copenhagen, 83 Solvgade, DK-1307 Copenhagen K, Denmark. Improvements in contrast and density of cellular constituents through the use of tannic acid (TA) during fixation or as a mordant between osmication and contrasting have been

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investigated in a wide variety of plant cells. Generally, as in animal cells, the effects range from increased osmiophilicity of protoplasmic membranes to conspicuous negative contrast in cytomembranes. TA and similar phenolics have similar effects when added experimentally during fixation, or leaching from native, intracellular sources which is a typical feature of many plant cells. In thylakoid membranes TA fixation/mordanting visualizes specific particles associated with the membrane (chloroplast coupling factor) and intramembranous complexes (large luminal photosystem II particles), and clearly resolves the lattice structure of crystalline inclusions. In neck regions of plasmodesmata in leaves and roots specialized, composite structures, possibly representing sphincters, are made visible after TA fixation. Observation of complex 26-nm granules associated with developing cell walls is fully dependent on the effect of TA; possibly these granules are involved in biogenesis of cellulose microfibrils. Optimal visualization of these specific macromolecules in plant cells was dependent on TA being added to the aldehyde fixatives rather than being applied after osmication. In TA-fixed cells the frequent appearance of fused membranes and high-contrast multilamellar bodies resembling phospholipid bodies in type II pneumocytes of animal lung tissue was noted. Control experiments with Zea mays germinated in tap water containing 1% TA have demonstrated (1) that germination and growth are promoted in the presence of TA and (2) high-contrast multilamellar bodies accumulate along plasma membranes of most cell types in root and shoot tissues. This indicates that the presence of TA during fixation under certain conditions can produce artifactual changes in membrane structure.

Visualization of Fc-Mediated Immune Precipitation. GUNNA CHRISTIANSEN AND N. P. H. MOLLER, Institute of Medical Microbiology, Bartholin Building, University of Aarhus, Denmark. The precipitating ability of isomolar solutions of intact rabbit IgG and F(ab')2 fragments shows that the Fc portion of IgG is of significant importance for the precipitation reaction. To visualize these immunocomplexes by electron microscopy a novel method has been developed. The method is composed of a three-step reaction carried out directly on the microscope grid. (1) Hemocyanin is adsorbed to carbon-coated grids. (2) The grids are then incubated in solutions containing antihemocyanin IgG or antihemocyanin F(ab'),, (3) Finally the grids are incubated in solutions of ferritin-antiferritin IgG or ferritinantiferritin F(ab')~. After the reactions have taken place, the grids are washed and negatively stained with uranyl acetate. It is found that only where Fc-Fc interaction has taken place, large complexes of ferritin molecules are seen around the hemocyanins.

Shrinkage in Preparatory Steps for SEM: A Study on Corneal Endothelium. J. U. PRAUSE, O. A. JENSEN, AND H. LAURSEN, Institute of Eye Pathology and Institute of Neuropathology, University of Copenhagen, Copenhagen, Denmark. Since specular microscopy of the cornea offers the opportunity to observe and measure cells in vivo without any outside interference this method forms an unrivalled basis for estimation of tissue shrinkage during various preparatory methods. Therefore a study was performed with the purpose of evaluating the degree of artifacts in each preparatory step from the living tissue in vivo to the final SEM specimen. The study was performed on rabbit corneas, the endothelium serving as measuring target. The in vivo state was recorded by specular microscopy. Unfixed corneas were studied by light microscopy unstained and stained by alizarin red S or silver nitrate. Fixation was performed intraca-

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merally with 1.5% glutaraldehyde (Gla) with pH, osmolarity, viscosity, and intraocular pressure identical to the physiological values of rabbit eyes. Fixation was completed by immersion in 2.5% Gla for V2 hr. Gla-fixed corneas were evaluated as above before osmification. Dehydration was performed either by graded acetone or by acetone in a gradient-free system, both followed by CPD. At all steps cells were counted using the same reference frame. The number of cells per square millimeter was estimated and statistical analysis showed a final areal shrinkage of about 30%.

Positive Identification of Cells by Light Microscopy (LM) prior to Scanning Electron Microscopy (SEM) Characterization. A. REITH, R. PUNTERVOLD, AND K. FEREN, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway. The identification of mitotic cells in culture by SEM criteria alone is generally not sufficient to distinguish cells in interphase from cells in mitosis. Usually cells in mitosis are identified by their "roundness" and microvilli-bearing surface in SEM. However, a certain amount of flat cells can be seen during mitosis which may account for nearly 50% in prophase. This can influence the results when recording long microvilli-bearing cells in cell culture exposed to carcinogens to screen for oncogenic transformation. An easy, non-time-consuming method is presented using Papanicolaou staining after fixation for SEM. After the staining the specimens are critical point dried, studied, and photographed in the LM. The specimens are then coated with gold and the same cells identified in the LM are taken up by SEM. This method allows study of SEM topography of cells in mitosis with precise determination of the mitotic phase.

Combined Light and Electron Microscopy in Routine Pathology. DAVID A. LEVISON, Department of Histopathology, St. Bartholomew's Hospital, London, England. A new type of transmission electron microscope, the combined light and electron microscope (LEM-2000), has recently been developed by International Scientific Instruments Ltd. (ISI). In the Histopathology Department, St. Bartholomew's Hospital, London, we have used a prototype LEM for 3 months, evaluating the instrument and associated techniques of specimen preparation. We have tested possible applications of t h e LEM to routine surgical histopathology. Initial details of the instrument suggested that it would be possible to observe the same specimen in both the light and transmission electron modes, combining selective colour staining with high-resolution microscopy in the electron mode. However, sections thin enough to produce good resolution in the electron mode were found to be suitable for light microscopy with a limited number of stains only. An alternative approach--using the instrument as a conventional transmission electron microscope, but exploiting the large grid size (7-ram diameter), low-magnification capacity in the EM mode (× 250), and the built-in microprocessor for recording areas of interest--has shown just what a useful instrument this can be in routine surgical pathology, overcoming sampling difficulties in a number of important pathological situations. Examples of work done on the instrument are illustrated and areas where the large grid size may be especially useful in histopathological diagnosis are discussed.

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Rotary Replication of Freeze-Fractured Na,K-ATPase. ELISABETH SKRIVER,ARVID B. MAUNSBACH, AND PETER L. JORGENSEN, Department of Cell Biology, Institute of Anat-

omy and Institute of Physiology, University of Aarhus, Aarhus, Denmark. Na,K-ATPase can be isolated from outer renal medulla in membrane-bound form. The isolated membranes contain, in addition to lipid, exclusively the proteins of the sodium pump. On the basis of particle frequency (about 6100//xm 2) and particle diameter (about 80-90 A) and the chemical and enzymatic composition of the membranes each intramembranous particle seems to correspond to one molecule of Na,K-ATPase with a molecular weight of about 280 000 and containing two large catalytic chains. This interpretation is supported by our observation that phospholipid vesicles reconstituted with purified Na,KATPase show similar intramembranous particles with a frequency proportional to both the amount of enzyme used in the reconstitution and the ion transporting ability of the vesicles. In the present study we have used freeze-fracture with rotary replication to further define the ultrastructure of intramembranous particles in purified ATPase membranes as well as in phospholipid vesicles reconstituted with Na,K-ATPase. Following fracture at temperatures ranging between - 105 and - 150°C the preparations were rotary replicated at angles between 10 and 45 °. When the shadowing angle was 10-15 ° and the amount of platinum evaporated was small most intramembranous particles in purified membranes and reconstituted phospholipid vesicles were slightly asymmetrical in shape and some particles appeared resolved in two subunits with a center-to-center distance of 35-45 A. The dimeric appearance of the particles was more frequent at -150°C than at higher temperatures. When the amount of platinum evaporated was increased the particles increased in diameter and acquired more heterogeneous shapes without evidence of substructures. The present observations are consistent with the interpretation that each intramembranous particle in the Na,K-ATPase preparations represents one enzyme molecule with a dimer substructure. In addition, the present observations indicate that rotary replication visualizes substructures not demonstrable by unidirectional shadowing and illustrate how structural details in the replica depend upon some of the technical parameters in the freeze-fracture procedure.

The Membrane Lesion of Complement. J. TRANUM-JENSEN* AND S. BHAKDI,t *Anatomy Department C, University of Copenhagen, The Panum Institute, DK-2200 Copenhagen N, Denmark, and tlnstitute of Medical Microbiology, D-6300 Giessen, West Germany. By methods of negative staining we identified the lytically active C5b-9 complex of human complement as a hollow, cylindrical macromolecule: 15 nm in height, internal diameter 10 nm, wall thickness about 1 nm. The cylinder is rimmed at one end by an annulus of 20-nm outer diameter. The terminus opposite the annulus possess an apolar surface enabling the cylinder to penetrate into a lipid bilayer. After membrane insertion, l0 nm of the cylinder, including the annulus, projects exterior to the membrane. The height difference of 5 nm between isolated and membrane-inserted complexes, together with the observation of increased permeability of liposomal membranes towards negative stains, indicate the formation of a transmembrane pore. Studies on rotationally and unidirectionally shadowed freeze-fracture replicas revealed the membrane-inserted portion of the complex on the fracture E face. In nonetched specimens the complex presents as a solid plug. Subjected to slight etching the plugs change to rings, confirming the presence of a central water-filled pore, deepened by the etching. Concomitantly, circular defects--

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hardly detectable in nonetched specimens--are revealed on the fracture P face, indicating escape of water through defects in the lipid monolayer. Association of the complex with native integral membrane particles seems only incidental by mere proximity. Deep etching reveals the extramembraneous portion of the complex on the extracellular surface of the membrane. Thus, freeze fracture studies corroborate the concept that the principal event in membrane pertubation by complement is the formation of a true transmembrane pore, walled by the inserted hollow, macromolecular C5b-9 complex.

Heteroduplex Analysis of Rat-Human Mitochondrial DNA. GUNNA CHRISTIANSEN AND CLAUS CHRISTIANSEN,Institute of Medical Microbiology, University of Aarhus, DK-8000

Aarhus C, Denmark. Mammalian mitochondrial genomes show some striking similarities. All mammalian mitochondrial DNAs (mtDNA) are double-stranded supercoiled covalently closed circles with a molecular weight of 107 daltons. To study evolutionary changes in mtDNAs we have analyzed the heteroduplex formations of mtDNA from rats and humans in various concentrations of formamide. The individual heteroduplex molecules show several welldefined regions of heterology. In each heterologous region the lengths of the two singlestranded branches always appeared identical in length. Comparisons of the heteroduplex molecules show that several larger parts have been conserved during evolution separated by regions of various heterology. They also show that inversion or duplication of DNA does not seem to be part of the evolutionary mechanism in mtDNA. The heteroduplex map has been aligned to the genetic map by using restriction fragments for the heteroduplex analysis. Thus this map can be compared to genetic maps.

Thyroglobulin Iodination--An Intracellular or Extracellular Process? RAGNAR EKHOLM, Department of Anatomy, University of G6teborg, G6teborg, Sweden. Autoradiographic observations after radioiodide administration in vivo indicate that the

site of iodination in the thyroid is the luminal side of the apical plasma membrane of the follicle cells. However, the presence of iodinated protein in isolated cells after incubation with radioiodide suggests that iodination is an intracellular process. The autoradiographic results have been questioned because H202, a necessary component in iodination, is considered to be generated in the cytoplasm. On the other hand, the location of iodoproteins in isolated cells has not been explored. We have now tried to elucidate these two problems. For the demonstration of H.,O., we utilized the cytochemical cerium technique on a system of isolated open thyroid follicles. Incubation with NADH or NADPH resulted in the formation of precipitate (cerium perhydroxide) which was closely associated with the luminal surface of the apical plasma membrane. No precipitate was seen along the basal plasma membrane. Inside the cells, reaction product was present only in endocytotic vesicles. KCN did not inhibit the reaction. No reaction product was found after incubation without NAD(P)H or with NAD(P)H + catalase. These findings indicate that H202 is produced in the apical plasma membrane. Preparations of isolated follicles and cells were incubated with radioiodide for 2 to 20 rain and prepared for autoradiography. Structurally well-preserved follicles, closed as well as open, showed the same labeling pattern as follicles labeled in vivo: silver grains over the follicle lumen close to the apical cell surface and no grains over the cells. However, parts of follicles with poorer structural preservation had a drastically different labeling: Silver grains were dispersed all over the cytoplasm and there were no grains along the apical plasma membrane. Well-preserved

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isolated cells showed no labeling, less well-preserved cells a rich labeling over the cytoplasm. These findings are interpreted to show that under close-to-physiological in vitro conditions the site of iodination is the apical surface of the follicle cells. Iodination in the cytoplasm occurs only in follicle cells showing signs of disturbed structural integrity.

Retention of Newly Synthesized Protein at the Apical Surface of the Thyroid Follicle Ceil. LARS E. ERICSON AND T. ()FVERHOLM, Department of Anatomy, University of GOteborg, Box 33031, S-400 33 GOteborg, Sweden. Newly synthesized protein (mainly thyroglobulin) is delivered to the periphery of the follicle lumen by exocytosis. Incorporation of iodine in both newly synthesized as well as " o l d " molecules of thyroglobulin also occurs in the peripheral part of the follicle lumen in relation to the plasma membrane of the follicle cells. Rats, pretreated with thyroxine, were injected iv with [3H]leucine or 125I and the thyroids were prepared for electron microscopic autoradiography. The distribution of silver grains over concentric regions of the lumen was determined. The distribution of radioactive label was calculated from the experimental grain distribution using data on resolution and considering the concentric regions as band-formed radioactive sources. In rats injected with [3H]leucine 90% of the luminal radioactivity was located in the microvillus region (average width 0.65/xm) after 3 hr, about 80% after 4.5 hr, and 60% after 6 hr. The remaining radioactivity was mainly present in regions adjacent to microvilli. After 1251 (30 min-2 hr) the radioactivity was spread in the lumen and a concentration to microvilli was less apparent. This indicates that an impaired diffusion did not cause the preferential location of newly synthesized protein to microvilli seen after radioleucine. The observations suggest that newly synthesized protein is selectively retained at the apical surface of the follicle cell.

The Time Sequence of the Subcellular Reaction Pattern of Peroxisomes in Rat Liver during Thyroid Hormone (T3) Administration. A Stereologic Study by Electron Microscopy. BIRGITTA FRINGES AND ALBRECHTREITH, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway. The subcellular reaction pattern of the rat liver cell has been studied following thyroid hormone administration at 1, 3, 5, 8, and 15 days (20/xg 3',3',5'-triiodothyronine, 100 g body wt/day). Mild hyperthyroidism caused by T~ was characterized by a significant parallel stepwise increase of the volume fraction of both mitochondria (77%) and peroxisomes (100%) up to the eighth day. The enlargement of the peroxisomal compartment, normally consisting of 31 × l0 s + 1.8 particles per 1 cm 3 liver cell cytoplasm, was achieved by a drastic biogenesis of organelles, which doubled after 1 day. This reaction was accompanied by a diminution of the average single particle volume (normally 27 + 0.02/zm 3) of about one-third (0.18 _+ 0.03 /zm3). The 100% increase of the peroxisomal fractional volume at 8 days was accompanied by a sixfold increase of the numerical density and a threefold decrease of their average single volume up to 0.08 _+ 0.02/xm 3. The marked increase in the number of peroxisomes was also reflected in their substructure, as indicated by a loss of profiles with the typical dense central core structure. Biogenesis of peroxisomes occurred at certain regions of the SER, as indicated by the increase of small peroxisomes (i) occurring in clusters and (ii) connections to the SER in the form of short stalks. Conclusion: The above-described peroxisomal reaction pattern under thyroid hormone influence may facilitate the peroxisomal function. In a peroxisome compartment consisting of more and conspicuously smaller organelles, the substrate availability for peroxisomal matrix enzymes may be improved by (i) an increase in the

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organelles' surface, (ii) a shortening of the substrate/enzyme distance within the organelle, and (iii) a closer proximity of mitochondria and peroxisomes. The corresponding reaction of mitochondria and peroxisomal volume fraction strongly implies the idea of a close metabolic interrelationship of the two organelle systems.

Effect of Long-Term Treatment with Pilocarpine on Secretory Granules of the Mouse Gallbladder Epithelium. R. HENRIKSSON, T. WAHLIN, AND H. AXELSSON, Departments

of Histology and Pathology, University of Ume~, S-901 87 Ume~, Sweden. A single intraperitoneal injection of pilocarpine significantly decreases the volume density of secretory granules in the principal cells of the mouse gallbladder mucosa. The effect of repeated pilocarpine injections on the gallbladder epithelium was studied. Adult male black mice were divided into four groups: (1) control animals; (2) animals decapitated 30 min after a single ip injection of pilocarpine; (3) animals treated with pilocarpine twice daily for 12 days (last injection given 24 hr before the animals are killed); (4) animals treated as group 3 but the last injection is given 30 rain before the animals are sacrificed. Randomly selected principal cells of the gallbladder epithelium were subjected to electron microscopic morphometric analysis. In animals treated with pilocarpine for 12 days the gallbladder principal cells increased in cell and nuclear size. The secretory-granule content was somewhat decreased in group 3 animals compared to the control group. The decrease of volume density of secretory granules 30 min after pilocarpine injection was less pronounced in animals treated for 12 days with pilocarpine. Repeated treatment with a cholinergic drug seems to influence the secretory behavior of glycoprotein granules of mouse gallbladder epithelium.

Localization of a Biotin-Binding Protein (Avidin) in Chick Oviduct by Immunoelectron Microscopy. I. RANTALA, H. HELIN, AND H. A. ELO, Departments of Clinical and

Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland. Avidin induction by progesterone in goblet cells in the oviduct of estrogen-pretreated chicks is an important model system in studying the mode of progesterone action. Intracellular localization of avidin is studied here by an immunoelectron microscopical procedure. Estrogen-pretreated chicks are injected with progesterone. One day later, small pieces of oviduct magnum mucosa are fixed in 4% buffered paraformaldehyde at 4°C for 6 hr. After washing the specimens in phosphate buffer, 40- to 100-/~m tissue chopper sections are prepared and incubated in goat anti-avidin serum at 4°C for 20 hr. Washed sections are incubated again in peroxidase-conjugated rabbit anti-goat IgG serum for 20 hr. Peroxidase activity is then revealed and the sections are treated with OsO4, dehydrated, and embedded in Epon. Avidin induced by progesterone administration is localized in the apical part of goblet cells and the plasma membranes of cilia adjacent to goblet cells. There is no avidin in goblet cells in chicks treated with estrogen only. The results indicate that immunoelectron microscopy is a powerful means of studying localization of a specific steroid hormone-inducible protein.

Effects of Long-Term Nickel Exposure on Rabbit Alveolar Epithelium. ANNE JOHANSSON*'t AND PER CAMNER,t *The Wenner-Gren Institute, University of Stockholm, No-

rrtullsgatan 16, S-113 45 Stockholm, and tThe Department of Environmental Hygiene,

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Karolinska Institute, and National Swedish Environment Protection Board, S-104 Ol Stockholm, Sweden. Rabbits were exposed to about 1 mg/m 3 respirable metallic nickel dust during 1, 3, and 6 months. Morphometric measurements on the lungs of the exposed rabbits showed about a twofold increase in the type II alveolar epithelial cells, compared to controls. In the exposed rabbits, some of the type II cells were extremely large, about 25-30 tzm in diameter compared to 10 tzm in the controls. These large cells were particularly common in the animals exposed for 3 months. The alveoli of the exposed lungs contained a yellowish amorphous substance, which has been shown to contain phospholipid. The amount of this substance increased with time of nickel exposure. Ultrastructurally the material consisted of lamellated structures and in the 6-month-exposed rabbits most of the alveolar spaces were entirely filled. The type II cells, which produce the pulmonary surfactant, seem to be stimulated in response to nickel exposure. This leads to an increased production of surfactant material which fills the alveoli to a degree where lung function may be impaired.

Are Alveolar Macrophages Translocated to the Lymph Nodes? ANNE JOHANSSON*"t AND PER CAMNER,t *The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16,

S-113 45 Stockholm, and tThe Department of Environmental Hygiene, Karolinska Institute, and National Swedish Environment Protection Board, S-104 O1 Stockholm, Sweden. Hilar lymph nodes from rabbits exposed for 3 months to 1 mg/m 3 respirable metallic nickel dust were examined. Some lymph node macrophages were filled with lamellated bodies, similar to those seen in the lung lavage fluid, the lung alveoli, and the cytoplasm of the alveolar macrophages after nickel exposure. In addition these lymph node macrophages had a vesiculated cytoplasm and well-developed endoplasmic reticulum and a few of them contained nickel particles as well. This means that they had the same appearance as the alveolar macrophages of exposed rabbits. We suggest that there is a transport of alveolar macrophages to the lymph nodes in nickel-exposed rabbits.

Is Secretory Membrane Recycled in Immunoglobulin-Producing Cells? PETER D. OTTOSEN,* PIERRE J. COURTOY,t AND MARYLIN GIST FARQUHAR,t *Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark, and #Section of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520. In secretory cells membrane is continually inserted in the plasmalemma during exocytosis of secretory product. In glandular cells it has been demonstrated that this is balanced by continual removal of membrane from the cell surface by endocytosis and that at least some of this membrane is transferred back to the Golgi cisternae. This indicates that the membrane might be reutilized in the formation of new secretory vesicles. In the present study we used cationic ferritin to follow the route of retrieved membrane in mouse myeloma cells which secrete large amounts of immunoglobulins in vitro. Three different cell lines were studied (x63 Ag 8, RPC 5.4, and MOPC 315). The cells were incubated in cationic ferritin for varying time intervals before fixation and preparation for electron microscopy. At early time intervals (2-10 rain) cationic ferritin was located in small vesicles in the periphery of the cells; at later intervals (10-20 rain) it was observed in increasing quantities in the Golgi region where it was located in smaller and larger vacuoles and in lysosomes. After 20 rain and later increasing amounts of cationic ferritin were observed in the stacked Golgi cisternae in two cell lines (x63 Ag 8, RCP

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5.4), but not in the third (MOPC 315). In separate experiments the location of secretory product (mouse immunoglobulin) was demonstrated using anti-mouse immunoglobulin Fab fragments coupled to peroxidase. When this was carried out on cells incubated in cationic ferritin it was shown that both cationic ferritin and secretory product were present in the same Golgi cisternae. Experiments on normal plasma cells from immunized rats also showed a vesicular transport of cationic ferritin from the cell surface to the Golgi cisternae. The main finding in the present study is the demonstration that membrane is continually removed from the cell surface and fuses with Golgi elements in immunoglobulin-producing cells, which suggests that the internalized membrane is reutilized in the formation of new secretory vacuoles.

Membrane Retrieval in Secretory Cells as Revealed by Electron-Opaque Tracers. V. HERZOG, Institut fiir Zellbiologie, Universitiit Miinchen, D-8000 Miinchen 2, West Germany. In secretory cells, the membrane of secretion granules is inserted into the cell surface during exocytosis and removed thereafter by endocytosis. In lacrimal and parotid glands, it has been demonstrated that part of the retrieved membrane is transferred to the Golgi stacks for possible reutilization during the formation of secretion granules (V. Herzog and M. G. Farquhar, 1977, Proc. Nat. Acad. Sci. 74, 5073-5077). Only a minor portion may also reach lysosomes. Little is known concerning the factors which may influence the pathway of retrieved plasma membrane. In rat exocrine pancreas, endocytic vesicles as traced with dextran (MW 44 000) rapidly reach Golgi cisternae, condensing vacuoles, and, later, mature secretion granules whereas horseradish peroxidase (MW 44 000) is shed exclusively into lysosomes. This suggests that in part the composition of the tracers may direct the route of endocytic vesicles (V. Herzog and H. Reggio, 1980, Eur. J. Cell Biol., in press). In epithelial cells of isolated thyroid follicles, native ferritin (NF) (pI 4.7) and cationized ferritin (CF) (pI 8.5) are transferred first to lysosomes. Later, however, CF is also found in stacked Golgi cisternae whereas NF remains within the lysosomal matrix. The results point to the importance of net charge of tracers on the fate of retrieved plasma membrane. Furthermore, it is suggested that the membranes of endocytic vesicles may detach again after their fusion with lysosomes and move forward to stacked Golgi cisternae.

Internalization of Cationized Ferritin into the Golgi Complex/GERL of Cultured Mouse Peritoneal Macrophages. JOHAN THYBERG AND JAN NILSSON, Department of Histology,

Karolinska Institutet, Box 60 400, S-104 O1 Stockholm, Sweden. Resident and thioglycollate-elicited mouse peritoneal macrophages were cultured in vitro and exposed to exogenous tracers in order to follow the fate of fluid and membrane internalized by endocytosis. Native, anionic ferritin and horseradish peroxidase did not bind to the plasma membrane and after being taken up were exclusively f o u n d i n endocytic vesicles and lysosomes. Cationized ferritin (CF), on the other hand, bound to the plasma membrane and was then rapidly ingested. Intracellularly, CF first appeared in endocytic vesicles and lysosomes, but was then also transferred to and passed through the Golgi complex/Golgi-endoplasmic reticulum-lysosomes (GERL). These observations indicate that content and membrane of endocytic vesicles partly may follow different paths within the macrophages. The content is emptied into lysosomes, from which part of the incoming membrane subsequently can be detached and moved over to the Golgi

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complex/GERL for reutilization and, eventually, recirculation back to the cell surface. Pretreatment of the cells with colchicine removed all cytoplasmic microtubules and caused a characteristic disorganization of the Golgi complex/GERL. The drug treatment did not affect the binding of CF to the plasma membrane, but inhibited its uptake and transport to the Golgi complex/GERL. Lumicolchicine was without effect on both surface binding and ingestion of CF. These findings support previous notions concerning a role of cytoplasmic microtubules in endocytosis.

Proximal Tubular Uptake of Anionic and Cationized Ferritin. ERIK I. CHRISTENSEN, FRANK A. CARONE, AND HELMUT G. RENNKE, Department of Pathology, Northwestern

University, Chicago, Illinois 60611, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark, and Department of Pathology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115. Although the renal handling of proteins in the proximal tubule cells has been intensively studied in recent years, the initial phases of the endocytic process, i.e., the binding of proteins to the luminal cell membrane are not well understood. The present experiments were performed to determine if the isoelectric point of a protein influences its endocytic uptake in the rat renal proximal tubule, as it is well known from studies with other cell types that positively charged proteins bind stronger to cell surfaces than do negatively charged proteins. Ferritin (MW - 480000, ae 61 /~) was used as tracer protein, the isoelectric point of the native (anionic) ferritin was 4.4 and that of the cationized ferritin 9.9. Either anionic or cationized ferritin was microinfused in vivo into rat surface proximal tubules for 30 to 60 sec. Three to four minutes later the same tubules were repunctured and microinfused with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer. The same fixative was then dripped on the surface of the kidney and small portions of the kidney containing the fixed tubules were removed and processed for electron microscopy. Nine tubules were infused with each type of ferritin, three tubules from each rat. The second segment of the proximal tubule was used for the analysis. Very little ferritin remained on the surface of the proximal tubule cells, but small amounts could still be seen in small apical vesicles and tubules. The ferritin was mainly located in large vacuoles either along the vacuolar membrane or dispersed throughout the vacuole. A quantitative analysis revealed that the uptake of cationized ferritin was eight- to ninefold greater than that of anionic ferritin. The results of the present experiments show that the molecular charge of a protein greatly influences its endocytic uptake by the proximal tubule cells. Thus, positively charged ferritin is reabsorbed much more effectively as compared to negatively charged ferritin, suggesting a stronger initial binding of the cationized ferritin to the luminal cell membrane.

Vesicular Membrane Flow from the Apical to the Basolateral Surface of the Choroidai Epithelium Visualized with Cationized Ferritin. B. VAN DEURS AND M. MOLLER, Institute

of Medical Anatomy, The Panum Institute, DK-2200 Copenhagen N, Denmark. Recent studies from our laboratory demonstrated that the choroid plexus epithelium absorbs hydrophilic molecules from the apical (ventricular) surface by micropinocytosis, and sequesters the molecules within the lysosomal apparatus. Moreover, these studies also indicated that a vesicular transport from the apical surface of the choroidal epithelium to the connective tissue and blood stream takes place (B. van Deurs, 1980, Int. Rev. Cytol. 65, in press). This was mainly based on the observation that after ventriculocis-

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ternal perfusion of HRP in physiologically well-controlled rats for 30 min or more, heavily HRP-labeled vesicles were distinctly fused with the basolateral epithelial membrane lining intercellular spaces without tracer or with tracer only immediately outside the opening of the tracer-labeled vesicle. H o w e v e r , transepithelial vesicular transport is a controversial issue, and results obtained with an enzymatic tracer with relatively low molecular weight like H R P are not b e y o n d question. A vesicular transport means that small areas of apical surface membrane are moved to the basolateral surface of the epithelium, a process which therefore can be followed ultrastructurally with a surface marker such as cationized ferritin (CF). After ventriculocisternal perfusion of CF in rats, membrane flow can be followed via micropinocytic vesicles to apical vacuoles and multivesicular bodies. In addition, CF labeling of vesicular profiles connected with the basolateral epithelial membrane or directly of the basolateral membrane or of the intercellular space were consistently observed. This could not be demonstrated with native (anionic) ferritin. With this high-molecular-weight polycationic membrane marker we exclude the possibility of penetration of the epithelial tight junctions or any kind of " r e t r o g r a d e " labeling (loc. cit.) of the intercellular spaces and bordering membranes, and thus conclude that a vesicular transepithelial transport most likely explains our observations.

Vesicular Transport of Intravenously Injected Microperoxidase across the Mouse BloodBrain Barrier. E. WESTERGAARD, Department of Anatomy C, Universitetsparken 1, DK-

2100 Copenhagen 0, Denmark. Previously it has been revealed by light microscopical examinations that intravenously injected dye stuffs (e.g., Evans blue or trypan blue) or Na-fluorescein are not able to cross the cerebral endothelium. The mentioned compounds bind to albumin. Later, horseradish peroxidase (HRP) was used as tracer, and it was demonstrated by electron microscopical investigations that a few, short segments of arterioles exhibited transfer of HRP across the endothelium. H R P (MW: 40 000) does not bind to albumin, and it is likely that the mechanism underlying the passage is vesicular transfer. Other tracers have been used, e.g., microperoxidase (MP, MW: 2000). It has been observed that MP, like HRP, is transported across the endothelium in short arteriolar segments (15-30/zm in diameter). No sign of interendothelial m o v e m e n t was obtained. The tight junctions might have functioned as a barrier to this small molecule. Some of the injected MP was bound to albumin, but part of the MP circulated freely in the bloodstream. It is also concluded for MP that the tracer might have been transferred by vesicles across the endothelium in a few segments of small arterioles of the brain. H o w e v e r , it was not possible to determine whether or not the amount of MP that crossed the endothelium was bound to albumin.

Unique Ultrastructure and Elemental Composition of the Stapedial Muscle of Guinea Pig. ROMUALD WR()BLEWSKI* AND MATTI ANNIKO,'~ *Wenner-Gren Institute, University of

Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, and ?Karolinska sjukhuset, S-104 O1 Stockholm, Sweden. M. stapedius is the smallest muscle of the mammalian body. By its contraction the input of sound especially of low frequencies into the inner-ear labyrinth is reduced. U1trastructural investigation showed an u n c o m m o n organization of stapedial muscle of guinea pig compared with that of its skeletal muscles or the stapedial muscle from other species. The cross section of individual muscle fibres was small (5-10 /zm (b) and the nuclei were centrally located. Golgi complexes and centrioles were frequently found near the nuclei. The mitochondria had a dense matrix and contained a large number of dense

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granules, adjacent to the internal membranes. Nerves were found among the muscle fibres and had end plates with extremely high amounts of vesicles. Capillaries appeared in a small number, approximately 1 per 30 muscle fibres. Degenerating and split muscle fibres were common. The muscle fibre composition based on its stainability for myofibrillar ATPase exhibited the presence of type I and type II fibres, the latter with subtypes A, B, and C. Elemental analysis performed on unfixed cryosections revealed an extremely low potassium concentration and a high concentration of calcium, sodium, and magnesium. The highest concentration of calcium and phosphorus was found in the mitochondria and their dense granules. The quadriceps muscle of guinea pig was also analysed as a control and its elemental composition was similar to that found in ordinary striated muscles from rat and man. Morphological and elemental composition features of guinea pig stapedial muscle are hence unique. Morphologically the stapedial muscle is reminiscent of myoblasts. The occurrence of centrioles and other cell components indicates a high metabolism. Low potassium and high calcium levels are difficult to explain, considering the function of calcium in contraction, its involvement in cell degeneration and death, and the function of potassium in maintaining the electrical potential difference between the inside of the muscle fibre and the extracellular medium. Regeneration of Rat Peritoneal Mast Cells after Histamine Release. ELLEN HOLM NIELSEN, PETER BYTZER, JORGEN CLAUSEN, AND NIRMAL CHAKRAVARTY,* Winslow Institute

of Human Anatomy and *Institute of Pharmacology, University of Odense, 5230 Odense M, Denmark. Regeneration of mast cells was followed in in vitro experiments from 10 sec to 48 hr after histamine release. Suspensions of mixed cells obtained by peritoneal lavage with a Krebs-Ringer solution were pooled from several rats and centrifuged. The cells were resuspended in Waymouth's medium with 1% BSA, Strep-Pen, and anti-PPLO, and incubated at 37°C. Samples were exposed to compound 48/80 for histamine release and fixed in 2.5% glutaraldehyde and postfixed in 1% OsO4. Sections for LM and TEM were stained with toluidine blue and uranyl acetate/lead citrate, respectively. Histamine release was determined by a fluorometric method. After incubation for 10 sec mast cells released most of their histamine by compound exocytosis. Granule matrices, however, were retained in several vacuoles inside the cell. The cell periphery consisted of a thin membranebounded cytoplasmic rim with scattered openings. From 10 sec to 2 hr the vacuoles tended to coalesce. Golgi vesicles occasionally contained progranules. The cell surface now presented long microvilli, which fused with the cell membrane in a pinocytosis-like manner. From 6 to 48 hr the peripheral rim grew thicker and contained many progranules and mature granules. The large vacuole decreased in size and its granule matrices stained darker red, indicating the presence of heparin. The long microvilli still formed pinocytosislike vacuoles, perhaps a method of decreasing the cell surface after the secretion. The fate of the granules inside the vacuole is presently unknown, as 48 hr so far is the longest survival time obtained for morphological studies of the regeneration of mast cells in vitro. Combined Light and Electron Microscopic Analysis of Physiologically Identified Neurons after Intracellular Deposition of Horseradish Peroxidase. J.-O. KELLERTH, S. CULLHEIM, AND P.-~. LAGERBA.CK,Department of Anatomy, Karolinska Institutet, S-10401 Stock-

holm 60, Sweden. The functional properties of single neurons have been recorded in anaesthetized cats with intracellular microelectrodes filled with a solution of horseradish peroxidase (HRP).

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After functional classification each neuron was iontophoretically injected with HRP. Following tissue fixation, processing for HRP, and section embedding, detailed light microscopic (LM) reconstructions could be obtained of the HRP-stained neurons, including their dendritic trees, axonal systems, and synaptic terminals. Any HRP-stained neuronal structure identified in the LM could also be further processed for ultrastructural investigation. The neuronal profiles containing the HRP reaction product were easily recognized in the electron microscope, and the presence of the reaction product did not seem to have affected the normal ultrastructure to any significant extent. The present technique has been successfully tested on motoneurons, interneurons, and primary afferents of the cat spinal cord, and it allows a close correlation between structure and function in single neurons, at both the light microscopic and the electron microscopic level. Normal and Postlesional Synaptology of the Medial Habenular Nucleus of the Rat. J.

ZIMMER, J. LAWRENCE,AND G. RAISMAN,Institute of Anatomy B, University of Aarhus, DK-8000 Aarhus C, Denmark, and Laboratory of Neurobiology, NIMR, Mill Hill, London, England. The habenulae of the rat are two symmetrical structures joined by the habenular commissure. The medial habenular nucleus contains both substance P and cholinergic neurons, projecting through the fasciculus retroflexus (FR) to the interpeduncular nucleus (IPN). Its main afferents arrive through the stria medullaris. A quantitative electron microscopic analysis of the IPN following sequential bilateral lesioning of the FR showed that the medial habenula on one side can reinnervate the synaptic sites in the IPN originally occupied by the contralateral habenula. We have now studied whether postlesional reactive reinnervation occurs in the medial habenula itself following lesions of the stria medullaris. First, the synaptic terminals of the medial habenula of normal adult rats were quantified and classified. Then, the normal and the degenerating synaptic terminals of the two medial habenulae were quantified from 2 days to 3 months after transection of the stria medullaris on one side. Finally, the same quantification was performed after the second stria was lesioned 3-6 months after the transection of the first one (sequential lesions). Two main types of terminals are found in normal medial habenula. One type (15% of the total population) forms symmetrical contacts with cell somas and straight dendritic shafts, contains vesicles with a tendency for flattening, and is surrounded by glia except on the side making synaptic contact. The other type forms intimate, complex relations with spines and more irregular dendritic structures. Tiny extensions from the spines (spinulae) often invaginate the terminals. The synaptic specializations are of the assymmetrical type, associated with aggregates of spherical vesicles, and many non-vesicle-associated, desmosome-like junctions are present. Unilateral stria lesions caused an initial decrease in both types of terminals in the ipsilateral medial habenula followed by a later increase to almost normal levels. While acute unilateral lesions did not provoke any contralateral terminal degeneration, sequential stria lesions did. This suggests that the primarily denervated habenula is reinnervated by strial fibers, which either normally cross in the commissure, but only terminate in the normal medial habenula to a very minor extent, or have grown through the commissure as a response to the denervation. A Quantitative Analysis of Axosomatic Boutons on Retrogradely Labelled Feline GraciloDiencephalic Relay Cells. ANDERS BLOMQVIST, Department of Anatomy, University of

Uppsala, Box 571, 751 23 Uppsala, Sweden. To study the synaptology of the gracile nucleus two adult cats were given thalamic

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injections of horseradish peroxidase. After a survival period of 48 hr, the animals were perfused transcardially with an aldehyde mixture. Transverse, 125-/xm-thick slices from the gracile nucleus were postfixed in osmium tetroxide and embedded in Epon following peroxidase processing. After polymerisation they were cut into 5-/xm-thick sections, which were mounted on acetate foil slides and examined in the light microscope. From the middle-dorsal part of the nucleus, 34 labelled neurons with a visible nucleolus were selected for ultrastructural analysis. The average diameter of these neurons ranged between 13 and 50 /xm. Ultrathin sections, which were cut from reembedded 5-/xm-thick sections, were contrasted with uranyl acetate and lead citrate and examined in the electron microscope. The soma membranes of the selected neurons were photographed systematically at a magnification of x 9800. Counts and measurements were made on prints (with a final magnification of x 20 000) by means of a graphic pen-data tablet device (MOP, Kontron). Most of the boutons contacting the soma membrane were s m a l l - t h e average apposition length of the bouton profiles was 1 . 5 / x m - - a n d contained small polymorphic vesicles. The bouton covering ratio (the proportion of the soma membrane covered by vesicle-containing profiles) varied considerably among the neurons and ranged between 1 and 63%. There was a statistically significant (P < 0.01) correlation between the size of the nerve cell body and the bouton covering ratio (r = 0.71). An analysis of 18 unlabelled neurons situated in the vicinity of the labelled ones showed a similar relationship. The results of the present study are in agreement with findings from other parts of the central nervous system that the larger neurons in general have higher values of the bouton covering ratio than the smaller ones. This relationship might reflect electrophysiological differences between large and small neurons.

A Stereological Approach to the Problems of Ultrastructural Scaling in Homologous Neural Structures. M. J. WEST, Anatomisk Institut B, Aarhus Universitet, 8000 Aarhus C,

Denmark. Stereological estimates of the density of synaptic contacts (number/ram 3) in the stratum moleculare fasciae dentatae of the mouse and rat made on electron micrographs indicate that there is no difference in the densities of these structures in the two species. Consequently, the absolute number of synaptic contacts is proportional to the volume of the stratum moleculare. Estimates of the number of granule cells with dendrites in the stratum moleculare indicate that the absolute number of granule cells is proportional to the 2A power of the volume of the stratum moleculare (i.e., proportional to the surface area of the stratum moleculare). If the mean number of synaptic contacts per unit length of dendrite is the same in both species, the dendrites must be longer in the rat than in the mouse and proportional to the ½ power of the volume of the stratum moleculare (i.e., proportional to the thickness of the stratum moleculare). The effect of increased dendritic length on the relationship between the volume and surface area of the granule cell dendrites in the two species will be examined with emphasis on the appropriateness of stereological approaches to neuroanatomical problems at the ultrastructural level.

The Effects of 4-Aminopyridine on Membrane Structures in Freeze-Fractured Mammalian Sympathetic Ganglia. C. FORSMAN AND L.-G. ELFVIN, Department of Anatomy,

Karolinska Institutet, Stockholm, Sweden. The ultrastructural effects of 4-aminopyridine on synapses and other membrane specializations in sympathetic ganglia of guinea pigs and rats were examined. When 4-AP

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was applied in a concentration of 1 mg/kg iv and ketamine was used to induce anesthesia the P face of the presynaptic membrane showed dimples or presynaptic membrane modulations (PMM) 30-45 nm in diameter. They were surrounded by large particles 11 nm in diameter. Such particles were also distinguishable inside the dimples. The synaptic area as a whole was densely populated with smaller particles. The presynaptic E face contained crater-like protrusions, which correspond to the PMM structures on the P face. Synaptic terminals in nontreated animals anesthetized with pentobarbital, 50 mg/kg ip, showed presynaptic membranes in which PMMs generally were more difficult to observe. In ganglia from guinea pigs exposed to 4-AP there also seemed to be an increase in the number of gap junctions. The gap junctions were all characterized by rows of particles separated by particle-free aisles.

Changes in the Volume Composition of the Neuropil in the Feline Lateral Cervical Nucleus during the Postnatal Development. R. FLINK, Department of Anatomy, Biomedical Cen-

ter, University of Uppsala, Box 571, S-751 23 Uppsala, Sweden. Earlier electron microscopical studies of the neurons in the lateral cervical nucleus (LCN) measuring the bouton covering on the soma in the nucleolar plane have shown an increase of bouton covering during the postnatal development. The aim of this study was to investigate this bouton increase further with stereological methods. The electron microscopical stereological examination was performed on 28 kittens perfused at different ages (12 hr, 2 days, 9 days, 15 days, 34 days, 92 days, and 120 days). Each age contained 4 kittens taken from the same litter. Ultrathin sections were cut from pieces of the LCN sampled at random and micrographs with the magnification of 8300 were taken at random using a coordinate system on the screen of the microscope and generating the coordinates with a table of random numbers. Using a point sampling screen with 168 points, the volume fractions of nine different parameters in the neuropil were calculated. The different parameters were: boutons, mitochondria in boutons, dendrites, mitochondria in dendrites, glial cells, myelinated axons, extracellular space, other identified structures (mainly blood vessels, unmyelinated axons, and neuronal perikarya), and other unidentified structures. Analysis of variance was performed and a probability of 0.05 was chosen as the significant level. The volume fraction of boutons increased during the early postnatal development from about 8 to 12%. A peak value of 14.5% was found at 34 days and the values then declined. These changes were found significant. The volume density of dendrites in the LCN was significantly declining in the observed ages. hi the newborn kitten, the dendritic fraction was estimated to 41% which decreased to 18.5% in the age of 120 days. The myelinated axons of course showed higher values during the postnatal development. The glial cells varied between 30 and 40%, however not significantly. Conclusions: There is a significant increase of the bouton volume in the LCN and it could be due to an increase both in the number and in the size of boutons. The decrease of dendrites during the postnatal development does not seem to have been demonstrated earlier. According to our results there is a relative reduction in the dendritic, volume of about 50% from birth to the age of 120 days and this decrease might indicate that the dendritic tree is initially overdimensioned to facilitate establishment of synaptic contacts.

Axon Diameter and Initial Myelination in Two Different White Matter Regions. S. REMAHL AND C. HILDEBRAND, Department of Anatomy, Karolinska lnstitutet, S-104 O1

Stockholm 60, Sweden.

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In the CNS the axons myelinate at a smaller diameter than in the PNS and the size ranges of unmyelinated and myelinated growing axons overlap considerably. In this study white matter regions, which undergo initial myelination at widely differing developmental stages, are compared in this respect. Prenatal (35-50 days gestation) and postnatal (1230 days) kittens were fixed by glutaraldehyde perfusion. Thin transverse sections were cut from the cervical spinal cord ventral funiculus (prenatal kittens) and from the corpus callosum (postnatal kittens). Unmyelinated, ensheathed, and myelinated axons were identified and measured on electron micrographs (× 10 000). The first myelin sheaths appeared 3 weeks before birth in the spinal cord and 3 weeks after birth in the corpus callosum. In the spinal cord the largest unmyelinated and the smallest myelinating axons measured 0.7-0.9 and 0.7-1.1 /~m, respectively. Corresponding callosal values were 0.5-0.7 and 0.4-0.6 ~m. Ensheathed but not myelinated axons measured 0.5-1.7/zm in the spinal and 0.4-0.9 /zm in the callosal specimens. The findings suggest that the diameter range at which axons first myelinate is markedly different in the two areas. This variability may be related to the developmental stage of the animal or to specific regional factors.

Preservation of Synaptic Vesicles in Cerebral Cortex Slices Incubated without Sodium Ions. R. I. PITKANEN, E. R. KORPI, AND S. S. OJA, Department of Biomedical Sciences,

University of Tampere, SF-33101 Tampere, Finland. Brain slices have often been used in studies on the release of synaptic neurotransmitters. Depletion of sodium from slices induces a great efflux of amino acid transmitters, and because the origin of this is at present unknown, we studied the content of presynaptic terminals by electron microscopy. Superficial rat cerebral cortex slices (0.5 mm thick) were incubated in Na-containing and Na-free (choline-substituted) Krebs-Ringer-Hepesglucose (KRH) medium, and thereafter fixed with 2.5% glutaraldehyde in N a - K R H and Ch-KRH medium, respectively, or in Na-phosphate buffer, all of which had rather similar osmolalities. Semithin sections displayed moderately well-preserved neurons and glial cells in the pial and the middle layers, but swelling and acidophilic large neurons near the cut surface of every slice. Phosphate-buffered fixative yielded the sharpest ultrastructure in slices incubated with or without Na. Presynaptic terminals with identified synaptic clefts were counted in areas 5/~m wide ranging from pial to cut surface. Slices fixed in phosphate buffer had about 13% terminals with less than 10 vesicles irrespective of the incubation medium. In slices incubated and fixed in N a - K R H and Ch-KRH " e m p t y " terminals amounted to 6 and 20%, respectively. The results thus suggest increased loss of vesicles during incubation without Na, although the preservation (or formation) of the vesicles also seems to depend on the ionic composition of the fixative vehicle.

A Light and Electron Microscopical Study of the Muscularis of Mouse Small Intestine: Interstitial Cell Types Associated with Plexus Muscularis Profundus. J. J. RUMESSEN, L.

THUNEBERG, AND H. B. MIKKELSEN, Anatomy Department C, University of Copenha-

gen, DK-2100 Copenhagen, Denmark. Plexus muscularis profundus (PMP) is situated between the two subdivisions of the circular muscle of muscularis externa of small intestine. Light microscopy (ZnIJOsO4) revealed PMP throughout the small intestine, with fasciculi forming elongated, circularly oriented meshes. Ganglion cells occurred widely scattered. Interstitial cells of Cajal (ICCIII) associated PMP were revealed as bipolar or stellate cells in a plexiform arrangement. ICC-III were selectively sought out by axons of PMP. Electron microscopy of PMP

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revealed: (1) nerve profiles containing numerous small (500 A) agranular vesicles together with a few large (1000 A) granular vesicles; (2) terminals containing numerous very large (1000-2000 A) dense-core vesicles together with few small (500 A) agranular vesicles. Nerve terminals were always separated more than 1000 A from muscle cells. Both types of nerve terminals formed synapse-like contacts to ICC-III, with a synaptic cleft of 100300 A. Presynaptic densities were frequently observed in endings containing predominantly small agranular vesicles. ICC-III exhibited basal lamina and caveolae with associated SER cisternae, and were interconnected, as well as connected to outer circularmuscle cells, by means of gap junctions. Also encountered were fibroblast-like cells dominated by moderately distended cisternae of granular endoplasmic reticulum. These showed no synapse-like contacts and no gap junctions. Processes of ICC-III occasionally enveloped macrophage-like cells, which contained conspicuous lysosomes.

A Light and Electron Microscopical Study of the Muscularis of Mouse Small Intestine: FITC-Dextran Uptake by Macrophages. H. B. MIKKELSEN, L. THUNEBERG, J. J. RUMESSEN, AND N. THORBALL, Anatomy Department C, University of Copenhagen, DK-

2100 Copenhagen, Denmark. Interstitial cells of the muscularis include apparently regularly distributed macrophagelike cells (MLC). Considering the known secretory functions of macrophages and the constant presence of MLC in the interval between the muscle layers we have commenced a study of the properties of MLC in an attempt to define subpopulations of these cells with respect to phagocytic and secretory activities. FITC-dextran (FITC-D) was used in order to study the phagocytic qualities of MLC. Albino mice were injected iv with FITC-D and killed by perfusion fixation 1, 3, and 6 days later. Specimens for fluorescence microscopy were embedded in Epon and 1-/~m sections were stained with Congo red. Specimens for EM were postfixed in either O s Q or K3Fe(CN)6-OsO4. By fluorescence microscopy abundant particles were found in cells fairly regularly distributed in the subserosal layer. Close to Auerbach's plexus (AP) the number of fluorescent cells was smaller and the cells contained fewer fluorescent particles. At the level of the plexus muscularis profundus (PMP) very few cells contained fluorescent particles. This distribution was confirmed by EM identification of dextran-containing vacuoles. In addition, dextran vacuoles in subserosal cells were larger compared to cells near AP.

TEM of the Langerhans Cells in Human Gingiva and Oral Mucosa. TOR ZELANDER, SVEND KIRKEBY,AND ERIK DABELSTEEN, Departments of Anatomy and Oral Diagnosis, The Royal Dental College of Copenhagen, Denmark. The well-established role of the Langerhans cell in contact dermatitis focused our interest on the same cell in allergic conditions of the oral mucosa. The aim of the present study is to establish the normal ultrastructure and distribution of the Langerhans cell in the human gingiva and oral mucosa as a foundation for functional and pathological investigations. So far biopsies from four individuals have been studied. After 2 hr fixation in cacodylate-buffered Karnovsky and 1½ hr in 1% O s Q the specimens were treated 30 rain in tannic acid followed by en bloc staining in 1% uranyl acetate, dehydrated in ethanol, and embedded in Epon. The thin sections were stained in 1% uranyl acetate in 50% ethanol and lead citrate. The cells are located in the basal two-thirds of the epithelium and the cell bodies with the indented nucleus are found one or two cell layers above the basement membrane. The long thin irregular dendritic processes are squeezed in the

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narrow spaces between the keratinocytes. The cytoplasm is clear, containing a few small mitochondria and one to two lysosomes. Desmosomes are absent as well as bundles of tonofilaments. The Langerhans granules--the EM marker of the c e l l - a r e most abundant in the Golgi region but may be found anywhere in the cytoplasm, often in contact with the plasma membrane. In a few instances the Langerhans cell has been observed in connective tissue underneath the basement membrane. The identity of the Langerhans cell is discussed with respect to ATPase activity of the oral epithelium and epidermal cells of the rabbit and guinea pig.

ATPase as Marker for Langerhans Cells. SVEND KIRKEBY, TOR ZELANDER, AND ERIK DABELSTEEN, Departments of Anatomy and Oral Diagnosis, The Royal Dental College

of Copenhagen, Denmark. Morphological identification of the Langerhans cell in TEM is made possible by the presence of the specific granules. In the light microscope conventional histological techniques are inadequate for demonstration of the cells. ATPase activity is one of the most convenient methods and is regarded as a very specific method of identifying the Langerhans cell. Biopsies from human bucca and gingiva, as well as slices from guinea pig and rabbit ears, were fixed for 4 hr by immersion in 4% paraformaldehyde buffered to pH 7.2. Cryostat sections were incubated in an ATPase medium with Mg2+ at pH 7.4. Tissue chopped sections were incubated in the same medium for TEM. In the light microscope enzyme activity was noted in some cells of the surface epithelia of human, as well as animal, origin. In human oral epithelium and guinea pig epidermis the reacting cells had an irregularly stellate shape, with thin drendritic processes projecting in different directions. Some of them extend close to the surface. In the rabbit ATPase-positive cells were different from the above. The cells formed a continuous basal layer, and had a regular cuboidal form. In the TEM the reaction product was located along the plasma membrane. The reacting basal layer of cells in the rabbit contained bundles of tonofilaments and rounded nuclei and desmosomes. No Langerhans granules were observed. Thus cells having a keratonocytic appearance show activity for ATPase. Accordingly the ATPase activity is not specific for the Langerhans cells in surface epithelia.

Electron Microscopic Study of Junctional and Oral Gingival Epithelia in the Juvenile and Adult Beagle Dog. LARS MATSSON, JORGEN THEILADE, AND ROLF ATTSTROM, Depart-

ments of Pedodontics, University of Lund, School of Dentistry, Maim6, Sweden, and Electron Microscopy and Periodontology, Royal Dental College, Aarhus, Denmark. Previous studies have demonstrated that there is a lower propensity to develop gingivitis in man and dog in the juvenile stage as compared to the adult stage. In juvenile dogs the junctional epithelium interposed between the gingival connective tissue and the tooth showed some resemblance to the keratinizing oral gingival epithelium, and a cuticular structure at the dentogingival junction suggested that the juvenile junctional epithelium might be less permeable to toxic substances eliciting inflammation. In the adult stage the oral gingival epithelium is considered to be less permeable than the junctional epithelium. As cytoplasmic filaments are held to be the main component in the process of keratinization and to have a stabilizing influence on the cells and tissues, the present investigation was designed to study the relative amounts of cytoplasmic filaments in the junctional and the oral epithelia of beagle dogs during juvenile and adult stages. The material consisted of gingival biopsies from six beagle dogs sampled when the dogs were 3 and 13 months

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old, respectively. On these occasions the gingiva was in excellent health. The biopsies were prepared for electron microscopic analysis and three randomly selected fields were recorded photographically from each of the following epithelial strata: basal and granular cell layers of the oral epithelium and basal and superficial cell layers of the junctional epithelium. Morphometric analysis was performed to estimate the density of cytoplasmic filaments of the cells in these epithelial strata. The amount of cytoplasmic filaments was considerably lower in the cells of the junctional epithelium than in those of the oral epithelium. In the oral epithelium the amount increased from basal toward superficial cells, whereas no such increase was seen in the junctional epithelium. The pattern was the same in the juvenile and the adult stage. Thus, the junctional epithelium in the juvenile stage does not have the characteristics of a keratinizing epithelium. Consequently other explanations must be sought for the lower gingivitis propensity in the juvenile stage. At the dentogingival junction all dogs had a thick, laminated layer of dental cuticle material in the juvenile stage. In the adult stage a similar structure was seen only infrequently, and never attained the thickness observed in the juvenile stage.

Life History of Pulpal Axons in Feline Primary Incisors. K. FRIED AND C. HILDEBRAND, Department of Anatomy, Karolinska Institutet, S-104 O1 Stockholm 60, Sweden. The early development and maturation of pulpal axons in primary teeth as well as the regressive changes in relation to shedding are of interest both from a general neurobiological and from a clinical point of view. Since the knowledge on these matters is highly incomplete the present study was undertaken. Decalcified mandibular incisors and extrapulpal incisor nerve branches from glutaraldehyde-perfused pre- and postnatal kittens were processed for electron microscopy. Ultrastructural observations were made on thin cross sections from the apical root canal and from the nerve branches. Intrapulpal unmyelinated axons were first observed 1 week postnatally. Myelination of these axons was initiated by 2 weeks. Two months after birth myelinated axons constituted 10-20% of all axons and ranged in size from 1 to 5 ~m. The myelin sheaths were disproportionately thin. At this stage a few unmyelinated axons presented signs of degeneration. Concomitant with onset and progress of root resorption an increasing proportion of unmyelinated and myelinated axons was affected. In late stages necrotic Schwann cells were seen related to degenerated axons, and there was a generalized pulpal tissue reaction. In some teeth where root resorption was advanced pulps totally devoid of axons were observed. A forthgoing derangement of all pulpal tissue elements continued until shedding, which took place at 3-4 months. Degenerative signs were not seen in extrapulpal incisor axons.

Progressive Involution and Physiological Cell Death of Smooth Muscle Cells in Pulpal Arterioles of Rat Incisors. N. THORBALL, H. MOE, AND H. WINTHER-NIELSEN, Anatomy

Department C, University of Copenhagen, DK2100 Copenhagen, Denmark. The rat incisor continues to grow and erupt throughout the life of the animal, being worn at the incisal end and replaced from the posterior (apical) end. Arterioles enter the pulp through the apical foramen and each of them supply a well-defined sector of the pulp. The pulp migrates with the tooth and the arterioles pass through a cycle of growth, remodeling, regression, and decay concomitant to changes in the pulp they supply. During the cycle mitosis of muscle cells and involution and death of muscle cells are observed in the arteriolar wall. The involution appears from the presence of segregations of cytoplasmic constituents, digestive vacuoles, and primary lysosomes with reaction of acid

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phosphatase and from signs of fragmentation. Cell death by early nuclear shutdown apparently without involvement of acid phosphatase degradation is also observed. These cells show conversion of polyribosomes into monoribosomes followed by cell condensation and fragmentation. Phagosomes with reaction of acid phosphatase are observed in small scale in the muscle cells and in large scale in periarteriolar macrophages. Thus, the arteriolar smooth muscle cells may go through a gradual involution by lysosomal degradation and fragmentation, or the cells may die in toto due to early nuclear shutdown. Small cell fragments may be removed by phagocytosis and digestion in neighbouring muscle cells. The bulk of the cell debris is removed by periarteriolar macrophages.

Rat Incisor Ameloblasts as a Model for Protein Synthesis and Enamel Secretion. H. WARSHAWSKY, Department of Anatomy, McGill University, Montreal, Quebec, Canada. All living cells synthesize "sedentary" protein for growth, metabolism, and turnover. Some cells, like the secretory ameloblasts of rat incisors, also produce specific "secretory" proteins which form extracellular layers. The ameloblasts were used in a radioautographic study to visualize the synthesis of these two types of proteins. After injection of [3H]tyrosine, an amino acid which is found in low amounts in enamel protein, the label was almost equally incorporated into proteins by nonsecretory and secretory ameloblasts. However, the incorporation of [3H]proline, which is prevalent in enamel protein, was highest in ameloblasts secreting enamel. Electron microscope radioautography of secretory cells showed reactions over the rough-surfaced endoplasmic reticulum at 2 rain after injection. Within 5 rain labeled proteins were in Golgi saccules and by 20 rain, labeled secretion granules were present. These granules were found at the two locations in the cell where secretion occurs: the cytoplasmic islands adjacent to the prong-like interrod growth regions between Tomes' processes, and the distal surfaces of Tomes' processes adjacent to rod growth regions. By 30 rain, labeling was seen extracellularly, but after several hours the newly deposited proteins appeared intermixed with previously secreted enamel. This "randomization" was inconsistent with the highly organized structure of enamel. However, correlation of radioautography with morphology revealed the following sequence: First, the initial layer of enamel is formed adjacent to dentin and this is the beginning of interrod enamel. Then, prong-like extensions form between Tomes' processes. These interrod prongs are produced as a collaborative secretion from the cytoplasmic islands and in three dimensions make up the walls of cavities occupied by Tomes' processes. The interrod cavities determine the shape and form of the enamel rods which are secreted progressively from one side of each Tomes' process until the interrod cavity is filled at the expense of Tomes' process. The initially deposited enamel proteins (amelogenins?) contribute to the lengthening of interrod and rod crystals, whereas the randomized proteins (enamelins?) may represent fragments of the original proteins which control the growth in width and thickness of the crystals. (Supported by grants from the Medical Research Council of Canada.)

The Development of Enamel Structure in Rat Incisors as Compared to Monkey and Human Teeth. H. WARSHAWSKY, K. JOSEPHSEN,* A. THYLSTRUP,* AND O. FEJERSKOV,*

Department of Anatomy, Faculty of Medicine, McGill University, Montreal, Quebec, Canada, and *Departments of Anatomy, Dental Pathology and Operative Dentistry, and Electron Microscopy, The Royal Dental College, Aarhus, Denmark. The rat incisor has repeatedly been shown to be an excellent model system in which

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to study the complex phenomena associated with amelogenesis. However, information obtained from this material has reluctantly been extrapolated to human amelogenesis because of the alleged structural differences between the teeth of these two species. The obvious differences include persistent eruption in rat incisors, as opposed to limited eruption in human teeth, and an enamel rod pattern in rats which seems to differ from the keyhole pattern, now well established for human enamel. Since these differences are emphasized by most workers in the field (see proceedings of the 3rd International Symposium on Enamel, 1979) it was felt that a comprehensive analysis of those features of enamel structure which are considered to be fundamental to the understanding of its formation, should be undertaken. The purpose of this study is to apply the detailed knowledge of amelogenesis as obtained in rat incisors to the teeth of monkey and man. The aim is to establish that there is a basic similarity between these species with regard to the existence of interrod enamel which forms depressions or pits occupied by Tomes' processes. Interrod enamel formation precedes the deposition of enamel rods within the confines of these pits. In addition, the rods form on one surface of Tomes' process and the accumulation of rod material compresses the process to one side of the pit resulting in arcade-shaped structures. Finally, the Tomes' processes do not withdraw from the pits, but are left as remnants which may either degenerate, or otherwise contribute to the accumulation of "nonenamel" material which in monkey and man may give rise to the arcade-shaped prism " s heat hs . " Such a comparative analysis demonstrates that it is no longer necessary to postulate a keyhole structure for primate enamel and it can be established that a direct similarity exists in the basic structures, and hence, in the mode of formation of the enamel in all these species.

The Production of Antibodies to Cell Membranes of Squamous Epithelium by the Use of Aldehyde-Induced Membrane Vesicles. ERIK DABELSTEEN, HENNING BIRKEDAL HANSEN, JYTTE WESTERGAARD, AND LISE FREDEBO, Departments of Oral Diagnosis, Oral Pa-

thology, Cariology, Periodontology and Electronmicroscopy, The Royal Dental College of Copenhagen, Denmark. A new method for the isolation of plasma membrane residues which can be used to raise antibodies to oral epithelial cell membranes is described. Plasma membrane vesicles were formed on the cell surfaces of confluent rat oral epithelial cell cultures by exposing the cells in situ to 100 mM freshly prepared formaldehyde. The vesicles formed 10-15 min after exposure and were released into the medium. The vesicles, 1-15/zm in diameter, were sedimented by centrifugation at 30 000g for 20 rain. Antibodies to vesicles were raised in rabbits and used in a double-layer immunofluorescence and immunoperoxidase staining technique. In rat and human oral epithelium they stained cell membranes in the basal and spinous cell layers. No cytoplasmic staining was seen in the epithelium. Staining of squamous epithelium from the human uterine cervix, rat and human skin, and guinea pig lip was negative.

Further Evidence of "Bleb" Formation in Striated Duct of Rat Submandibular Gland. E. B. MESSELT AND E. DAHL, Department of Anatomy, Dental Faculty, University of

Oslo, P. box 1052 Blindern, Oslo 3, Norway. Salivary gland striated duct cells are usually characterized morphologically by the presence of numerous radially arranged mitochondria and infoldings of the basal plasmamembrahe. The apical cytoplasm contain vesicles, granules, and microvilli. Structural alter-

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ations consisting of large bleb-like projections of cytoplasm into the ductal lumen have been reported several times. Their nature and origin have been debated. The purpose of the present investigations was to collect further evidence of the nature of bleb formation. The material employed in this work consists of rat submandibular glands perfused with 2.5% glutaraldehyde, postfixed in osmium tetraoxide and embedded in Vestopal-W. The blebs were of about the same density as the ground cytoplasm, but appeared lighter due to lack of prominent cytoplasmic organelles. The basal part of the blebs showed numerous ruptures which were present in both TEM and SEM examinations. Judged by the presence of membranous debris in the lumen, it was apparently the fate of these blebs to be separated from their cells of origin and to disintegrate. The blebs thus discarded result in rudimentous wail-like projections adhering to, and encircling the luminal surface of the cells. It appears that the cells rapidly regain a microvillous surface.

An X-Ray Microanalytical Study of Calcium Compartmentalization in Calliphora Salivary Glands. TUDOR BARNARD,1 THEODORE HALL, AND BRIJ GUPTA, The Biological Micro-

probe Laboratory, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, England. Using cryopreparative methods and an X-ray microanalysor equipped for the quantitative analysis of thin biological specimens, we have investigated the compartmentalization of calcium in resting, stimulated, calcium-depleted, and damaged Calliphora salivary glands. In thin ( 2 1 /xm), frozen-dried sections sandwiched under a Formvar film, mitochondria, nuclei, canaliculi, and basement membrane were recognisable. Analyses were made on mitochondrial profiles and adjacent areas of "nonmitochondrial cytoplasm." For calcium, no large differences were detected, levels generally being 5-10 raM/ kg dry wt in both resting and secreting glands. In a few observations on calcium-depleted glands, mitochondrial calcium levels were close to the minimum quantifiable ( - 0 . 5 mM/ kg dry wt) and nonmitochondrial cytoplasmic calcium was too low for quantification. High levels of mitochondrial calcium (~50 mM/kg dry wt) were found only in damaged cells of resting or secreting glands. These results show that the mitochondrial calcium levels in intact cells of Calliphora salivary glands are 10- to 100-fold lower than the levels predicted for various tissues on the basis of in vitro studies of isolated mitochondria. On the other hand, the results are in agreement with other recent microprobe data on muscle mitochondria. Further observations are required to elucidate if the mitochondrial calcium pool is directly used to control fluid secretion in the Calliphora salivary gland.

Transmission Electron Microscopy in Diagnostic Virology. ELISABETHKJELDSBERGAND B. V. JOHANSEN, National Institute of Public Health, Oslo, Norway. Direct and immune electron microscopy have gained an important role in diagnostic virology as alternative and supplemental methods to conventional virological techniques. In some cases they may even be the only usable approach. The advantages of EM compared to other available methods in virology are the speed, the lack of specificity, and the possibility to visualize viruses that are difficult or impossible to culture. Since most virus particles have sizes (20-500 nm in diameter) below the detection limit of the light microscope, electron microscopy is the only choice for visualization. In spite of a variety of different viruses they are divided into three main morphological groups comprising 1 Present address: Wenner-GrenInstitute, Norrtullsgatan 16, S-113 45 StockhoLm,Sweden.

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viruses with (i) icosahedral (cubic) symmetry, (ii) helical symmetry, and (iii) complex structure. Finer details of inner and outer structures of the particles are the basis of further subgrouping. The majority of viruses usually found in clinical specimens, e.g., herpes, adeno, pox, influenza, parotit, have a well-known and defined structure. There are, however, still viruses discovered in association with human diseases, whose morphological characteristics require some clarification before they can be placed in a structural group. Negative staining is the most suitable contrasting technique in viral diagnostic work. As an all-around stain 2-3% phosphotungstic acid (NaPT or KPT, pH 6-7) may be used since most viruses are well stained at these conditions. Thin-sectioning technique of embedded specimens is not a routine method in viral diagnosis due to a complicated and time-consuming methodology. Nevertheless in special cases, as for instance with brain material where the great lipid content makes it almost impossible to use negative staining, ultramicrotomy is recommended. Clinical specimens like crusts and vesicular and pustular fluids can be stained directly. Other specimens like feces, urine, spinal fluid, blood, biopsy material, nasopharyngeal, and eye secretions and washings may, due to their impurity and low concentration, be pretreated by extraction and ultracentrifugation before negative staining. Immune electron microscopy (IEM) is a useful method in the subtyping of a virus within a morphological group and, performed on serumpair and virus containing specimens, it may demonstrate an antibody response which suggests that the virus is the etiological agent of the disease. IEM is also suitable in attempts to find small viruses with uncharacteristic structure and in low concentration, since aggregates are easier to detect than single particles.

Analysis of Integrated Viral DNA in an Adenovirus Type 2 Transformed Rat Cell Line F19. GUNNAR WESTIN, JANUSZ ZABIELSKI, AND ULF PETTERSSON, Department of Mi-

crobiology, The Biomedical Centre, Box 581, Uppsala, Sweden. The human adenovirus type 2 is able to transform cells from newborn rats and hamsters, and it has been shown previously that as little as 14% from the left-hand end of the genome is present in these cell lines. The Fl9 genome was first cleaved with EcoRI followed by hybridization with nick translated a2P-labeled Ad2 DNA using the Southern blot technique. It was found that viral sequences were present in approximately 20-kblong EcoRI fragments. Using the Charon ~ vector 4A, it was possible to ligate the appropriate fragment from cell line F19, together with the left and right arms of the vector. In vitro packaging was done, followed by transfecting E. coli strain DP50 with the recombinant phage DNA. Some 100 000 recombinants were screened for the presence of viral sequences using the plaque hybridization method of Benton and Davis. From 33 positive recombinant phages, two clones, 1A and 2B, were further analyzed by hybridization and electron microscopy. Further Southern blotting on the two clones indicated the presence of Ad2 sequences mapping exclusively in restriction fragment SmaI-E (2.8510.7 map units) and XbaI-E (0-3.85). Heteroduplexes were constructed between the vector and clone 1A and 2B, to obtain the size of the cloned DNA, 16.4 and 17.2 kb, respectively. Using the restriction fragment Sinai-E, we constructed heterotriplexes to identify the position of the integrated sequences. From 25 heterotriplexes of each clone, the area of homology was estimated to be 100 nucleotides and was located 140 base pairs from one end of the SmaI-E fragment. Up to three SmaI-E fragments hybridized to a specific part in the longer arm of the substitution loop of the heterotriplex molecule.

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The Reliability of Electron Microscopic Observations in the Classification of Glomerulonephritis. S.-O. BOHMAN, H. J. G. GUNDERSEN, A. B. MAUNSBACH, AND T. STEEN

OLSEN, Department of Pathology, Karolinska Instituter, Huddinge Hospital, Huddinge, Sweden; Department of Cell Biology, Institute of Anatomy, and Institute of Experimental Clinical Research, University of Aarhus and Department of Pathology, Kommunehospitalet, Aarhus, Denmark. A systematic blind, semiquantitative electron microscopic procedure was developed for the ultrastructural analysis of human renal biopsies in glomerulonephritis. Different glomerular lesions were evaluated semiquantitatively using a 4° scale. The method was applied to biopsies from 81 consecutive glomerulonephritis and 11 kidneys without glomerulonephritis. Different parameters for the reproducibility of semiquantitative scoring, representativeness of micrographs, and representativeness of glomeruli were calculated. The reliability of the method was considered good or acceptable with respect to 17 out of 34 different ultrastructurally defined lesions including different types of "electrondense deposits," mesangial widening, and foot process retraction. For l0 of the lesions, e.g., crescent formations and peripheral mesangial interposition, the scoring was less useful due to a focal and/or segmental distribution of the lesions. The present data are relevant when evaluating the significance of ultrastructural findings in biopsies and for the assessment of the amount of ultrastructural data necessary for reliable judgements (S.-O. Bohman, N. Deguchi, H. J. G. Gundersen, J. Hestbech, A. B. Maunsbach, and S. Olsen, Lab. Invest. 40, 433, 1979). In order to assess the value of electron microscopy in diagnostic work the ultrastructural lesions in individual biopsies were also compared with the changes observed by light microscopy. A good correlation between the light and electron microscopical observations was found for several lesions, e.g., mesangial widening and mesangial hypercellularity while others, such as "deposits" of various types in the capillary wall were more reliably evaluated in the electron microscope. Crescent formations and adhesions between capillary tuft and Bowman's capsule are examples of lesions which were better evaluated in the light microscope. When the light microscopical biopsy findings were used to classify glomerulonephritis into different types according to a commonly used classification system, the ultrastructural data showed that several of the classes appeared heterogenous with respect to the type of pathogenetic mechanisms involved.

The Quantitative Relationship Between Foot Process Width and Proteinuria in Glomerulonephritis. TORBEN SEEFELDT, SVEN-OLOF BOHMAN, HANS JORGEN G. GUNDERSEN, ARVID B, MAUNSBACH, AND STEEN OLSEN, University Institute of Pathology, Kom-

munehospitalet, Department of Cell Biology, Institute of Anatomy, and Institute of Experimental Clinical Research, University of Aarhus, Aarhus, Denmark. The foot process width was analyzed in consecutive material of patients with glomerulonephritis defined by clinical criteria. A stereological method is used to obtain the distributions of true width of foot processes from that of observed apparent width on electron micrographs. The harmonic mean foot process width was related to the diurnal protein excretion, for which retrospective data were available in 79 of the patients. For the material as a whole only a moderate degree of correlation was found between foot process width and proteinuria, rank correlation ~- = 0.40, 2/' ~ 0.01. This was also the case in the 17 patients with "minimal change" disease, ~- = 0.53, 2P < 0.05, 8 of whom had normal foot process width. Finally, the relationship between the quantitative and a

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semiquantitative method for estimating foot process width was analyzed, showing a good correlation, ~- = 0.7, 2P ~ 0.01. In conclusion, foot process width does show a relation to proteinuria in glomerulonephritis, but only to a moderate degree. The semiquantitative evaluation of foot process width--which is a very fast procedure--appears to be a precise method for clinical studies.

Autoradiographic Studies of the Thymidine Incorporation in Diabetic Hypertrophic Kidneys. R. RASCH AND J. O. RYTTER NORGAARD, Department of Cell Biology, Institute of

Anatomy, University of Aarhus, Denmark. Incorporation of thymidine has been studied in streptozotocin diabetic uninephrectomized rats in order to see which parts of the nephron are involved in the diabetic kidney hypertrophy. Female Wistar rats weighing 120 g were given 1 mCi/kg of tritiated thymidine intraperitoneally 2, 4, and 6 days after the induction of diabetes. One hour later the kidneys were fixed by perfusion and embedded in Epon. Both 1-/zm-thick and ultrathin sections were cut and processed for autoradiography at light and electron microscope level. Three sections of cortical tissue have been studied in 7 animals from each group. The thymidine incorporation, expressed as percentage labeled nuclei, was measured in proximal tubules, distal tubules, and glomeruli. The studies showed that the kidney weight was approximately 40% increased in the diabetic animals after 2 days compared to controis. In both the proximal and the distal tubules the number of labeled nuclei was significantly increased (P < 0.01) after 2 days of diabetes when compared to controls. In the proximal tubules 8% of the nuclei were labeled, in the distal tubules 6%, and in controls around 1.2% in both proximal and distal tubules. In the following days a decrease in the incorporation of thymidine was seen. In glomeruli, however, the thymidine incorporation was decreased after the induction of diabetes, but the difference was not significant compared to controls. The glomerular labeling was low, around 1% or less, in both controls and diabetic animals. The present study has demonstrated that the previously reported enlargement of glomeruli seen after short-term diabetes is due to a hypertrophia of the cells, whereas an increased thymidine incorporation in the tubuli of the diabetic animals indicates that cellular hyperplasia is partly responsible for the kidney growth in these parts of the nephron.

Stereological Estimation of Glomerular Morphological Structures in Experimental Diabetes Mellitus Using a Combination of Light and Electron Microscopy. O. GOTZSCHE, H. J. G. GUNDERSEN, AND R. ~STERBY, Institute of Pathology and Department of Cell

Biology, Institute of Anatomy, Aarhus University, Denmark. The effect of islet transplantati~.n on the glomerular changes in streptozotocin-treated Lewis rats was studied. Weight-, Sex-, and age-matched animals were allocated to three groups, Normals, Diabetics, and Transplanted. Kidneys were perfusion fixed in situ under standardized conditions and cut into a series of slices of alternating thickness (3 and 0.8 ram). Three sections from each thick slice were PAS stained and used for the light microscopic determination of glomerular volume fraction. From the thin slices sections were cut from three strictly randomly selected glomerular cross sections which were photographed in toto (3200 ×, low-magnification electron micrograph, LMEM). A systematic independently positioned sample comprising an average of 10 micrographs per cross section was photographed (19 500 ×, high-magnification electron micrographs, HMEM). Assuming a specific gravity of kidney structures of 1 g/cm ~ the total glomerular volume

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in the left kidney V (glom) equals Vv (glomeruli/kidney) × kidney weight. On LMEM micrographs the individual tuft volume fractions Vv (tuft/glom) was estimated by point counting using the glomerulus as reference space. On the HMEM the fractional volumes of epithelium, endothelium capillary lumen, mesangial region, and peripheral basement membrane (PBM) were estimated by point counting with the tuft as reference volume. By multiplication the absolute individual quantities were obtained, e.g., V (PBM) = Vv (peripheral BM)/tuft x Vv (tuft/glom) × V (glom) mm 3. The study showed a 25% increase in the total amount of PBM material (from 0.85 + 0.05 (SEM) mm 3 to 1.07 ___0.04) after 4 weeks of diabetes and that islet cell transplantation followed by 4 weeks of normoglycemia had little effect on this parameter (1.05 _+ 0.07).

Nephrotoxicity of the Mushroom Cortinarius speciosissimus in the Rat. Y. HIRSIMAKI,* A. LAASONEN,* P. HIRSIM~.KI,* AND L. NIEMINEN,~" *Department of Cell Biology, Uni-

versity of Jyviiskylii, SF-40100, Jyviiskylii 10, and tFarmos Group LTD, P.O. Box 425, SF-20101 Turku 10, Finland. Cortinarius speciosissimus, a toxic mushroom, induced several fatal intoxications in Finland at the beginning of the 1970s. An electron microscopical (EM) study was made using rats in order to further elucidate the toxicity of this mushroom. Results were compared to light microscopy (LM) and physiological investigations on renal functions. Dried mushroom was fed to rats in water homogenate as a single dose. Samples for EM and LM were taken from the cortical and medullary areas I, 2, 3, and 6 days after administration. Physiological parameters indicated severe intoxication at a dose of 1500 mg/kg. The weight of the kidneys and urine osmolarity were increased 1 day after administration. Urine volume, concentration of proteins, glucose, and fl,_,-globulines, and ASAT and L D H activity were increased and excreted Na + and K+-ions were increased after 2 days. Acid phosphatase was decreased in plasma after 1 day. Alcalic phosphatase and L D H were increased after 3 days. At a dose of 700 mg/kg the corresponding effects were slight and were not observed at the lowest dose of 350 mg/kg. After 3 days inflammatory infiltrates were found in LM samples in the medulla near the cortical border. Necrosis was found in some proximal convoluted tubuli. After 6 days the number of inflammatory cells was reduced and scar formation was found. A large proportion of the proximal and some distal tubules were dilated and had flat and atrophic epithelia. Dilated collecting ducts contained inflammatory cells and cellular debris. In electron microscopical samples tubular cells and their nuclei were swollen. Chromatin margination and pycnotic nuclei also were observed. The endoplasmic reticulum was vesiculated and the amount of free ribosomes was increased. After 3 days the changes were more obvious. In addition smoothsurfaced ER was proliferated in proximal tubular cells and the intracristal space of mitochondria was swollen. Some necrotic cells were found. Glomeruli were normal both in LM and EM samples. We conclude that Cortinarius speciosissimus induces a dose-dependent delayed nephritis mainly in proximal tubuli.

Post-mortem Electrolyte Redistribution in Rat Studied by X-Ray Microanalysis and Electron Microscopy. GEMMA A. J. KUYPERS, ROMUALD WROBLEWSKI, AND GODFRIED M. ROOMANS, Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-11345

Stockholm, Sweden. A number of diseases is associated with abnormal elemental distribution at cell and tissue level. X-Ray microanalysis is hence a potentially useful technique in pathology,

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but its use in autopsy examination requires knowledge about possible artifacts. In the present study, postmortem electrolyte redistribution in various tissues from rat was studied, and correlated with morphological changes in these tissues. Pancreas, liver, cardiac muscle, and quadriceps femoris muscle were removed from the animal immediately after death, or after keeping the dead animal for 2 or 4 hr, respectively, at room temperature. Elemental distribution at the cellular level was studied by X-ray microanalysis of thick cryosections. Calcium redistribution at the subcellular level was studied using tissue fixed with glutaraldehyde/oxalate. In all tissues, postmortem redistribution of electrolytes had taken place within 2 hr. The cellular concentrations of Na, C1, and Ca increased markedly, those of Mg and K decreased; no significant changes were found in the concentrations of P and S. The number of oxalate precipitates (indicating the presence of calcium) increased both in the mitochondria and in the cytoplasm and endoplasmic reticulum. Morphological changes included mitochondrial swelling and vesiculation of the endoplasmic reticulum. Since the postmortem ion shifts are similar to those encountered in some diseases, great care has to be taken in the interpretation of X-ray microanalytical results from autopsy material.

Electrolyte Redistribution in Cystic Fibrosis Fibroblasts Studied by X-Ray Microanalysis and Electron Microscopy. GODFRIED M. ROOMANS,* OVE CEDER,t GEMMA A. J. KUYPERS~* A N D HANS KOLLBERG,t *Wenner-Gren Institute, University of Stockholm, Norr-

tullsgatan 16, S-11345 Stockholm, and tPediatric Clinic, Umed University Hospital, S90185 Umed, Sweden. Malfunctioning of ion-transport processes in secretory glands of cystic fibrosis (CF) patients may be a common factor underlying the diverse clinical symptoms of CF. Although fibroblasts are not exocrine cells, they are classically secretory cells, and for practical reasons suited as a model system to study CF. The elemental distribution in cultured fibroblasts of CF patients was compared with that in fibroblasts of age-matched controls. The cells were grown to near-confluence in plastic film-dishes, shock-frozen and freeze-dried. To some cultures Staphylococcus aureus protein A and human IgG were added. The cells were viewed in the SEM and single cells were analyzed by X-ray microanalysis. In addition, the distribution of calcium was studied in TEM after fixation with glutaraldehyde/oxalate. Fibroblasts of CF patients contained significantly more Ca (P < 0.02) and less Na (P < 0.05) than control fibroblasts. Addition of protein A and IgG did not influence the elemental composition of the fibroblasts. Morphologically, little difference could be observed, but a somewhat higher incidence of pinocytotic vesicles in CF fibrobIasts was noted. Our data may point to a connection between redistribution of Ca in cells of CF patients and the known malfunctioning of Na transport in exocrine glands of these patients, and offer new directions for studies of the basic defect in CF.

The Possible Role of Endothelium in the Control of Connective Tissue Repair in Arterial Tissue. J. CHEMNITZAND B. COLLATZCHRISTENSEN, Winslow Institute of Human Anat-

omy, University of Odense, DK-5230 Odense M, Denmark. The neointimal hyperplasia following a severe mechanical lesion of the rabbit thoracic aorta was studied by vital staining with Evans blue and transmission electron microscopy of specimens en bloc stained with tannic acid. The specimens were cut perpendicularly and tangentially to the surface of the vessel. Twenty-one days after lesion difference in the composition of connective tissue related to endothelium and pseudoendothelium

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(smooth muscle cells, SMC) was obvious. Neointimal connective tissue covered with endothelium contained fibronexus-like attachment fibers (an orthograde continuity between intracellular microfilaments and extracellular microfibrils across dense regions in the plasma membrane) connecting endothelial cells with subendothelial elastin. Incomplete elastic lamellas built up from elastic grains knitted together by microfilaments and glycosaminoglycans (GAG) were oriented parallel to the endothelial surface around myointimal cells. The content of collagen fibers was sparse. Neointimal connective tissue covered with SMC was poorly organized. Coarse elastic grains were randomly dispersed, the content of GAG was sparse, and collagen fibers were prominent. Apparently there was a defect in the formation of lamellar elastic tissue in which GAG and microfibrils seemed to play a role. Although the endothelial barrier function alone may sufficiently explain the influence on the course of healing processes, our results indicate an interaction between endothelial cells and SMC.

Quantitative Morphometry of the Diabetic Pancreas. J. P. KROUSTRUP, Institute of Anatomy C, Institute of Pathology and Second University Clinic of Internal Medicine, Aarhus University, DK-8000 Arhus C, Denmark. The total volume of the islets cells was measured in normal dogs and in dogs with 4day streptozotocin diabetes. After perfusion fixation with 1% glutaraldehyde and 1% paraformaldehyde, the whole pancreas was cut into equidistant sections and embedded in paraffin for LM and in Epon for EM. From each section the total islet volume was measured by LM point-counting on randomly selected equally spaced fields of vision and demonstrated an increasing islet volume from the uncinate to the lienal part of the pancreas. This pattern was seen in both groups; however, with a reduced islet volume in the diabetics (577 _+ 180 mm 3 versus 324 +_ 50 mm3). From each section one whole islet profile was selected at random and photographed in the EM at low magnification. Based upon the ultrastructure of the secretion granulas, the four endocrine cell types were quantitated by point-counting. An increasing A-cell volume and a decreasing PP-cell volume from the uncinate to the lienal part of the pancreas were observed in both groups, but only the B-cell total volume showed a significant decrease in the diabetic animals.

Ultrastructure and Cytochemistry of Adenoid Cystic Carcinoma. BENGT CARLSOO,GUNNARD. BLOOM, AND Slw DOMEIJ, Departments of Otorhinolaryngology and Histology,

University of Umed, Umed~, Sweden. Adenoid cystic carcinomas of salivary gland origin were studied using electron microscopic cytochemical techniques. At the ultrastructural level the replicated basement lamina of the pseudocysts, a prominent entity of these turnouts, displayed a strong positive reaction with the Pa-CrA-silver staining technique. Furthermore, amorphous material within the lumina of small duct-like structures also displayed a positive reaction. The amorphous material of the cystic compartments was less reactive. Numerous matrix granules present within the pseudocysts were furthermore positively stained with ruthenium red, whereas the replicated basement lamina remained unreactive. The matrix granules of the pseudocysts markedly resemble the extracellular polygonal, nonmembranebound granules found within hyaline cartilage. The present EM studies together with previous light microscopic histochemical investigations indicate that the matrix granules contain sulfated proteoglycans. Thus the histochemical as well as submicroscopical resemblance between benign mixed turnouts and adenoid cystic carcinomata is striking.

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Ultrastructure of the Basement Membrane Matrix Deposited by Parietal Yolk Sac Carcinoma Cells. ILMO LEIVO*'t AND JORMA WARTIOVAARA,* Departments of *Electron

Microscopy and tPathology, University of Helsinki, Helsinki, Finland. Mouse parietal yolk sac carcinoma (PYS-2) cells, derived from a differentiating teratocarcinoma line, secrete and deposit abundant basement membrane matrix. The amino acid composition of this material closely parallels that of embryonic parietal yolk sac basement membrane. Our metabolic labeling studies indicate that isolated PYS-2 matrix contains only two high-molecular-weight glycoprotein components, type IV collagen and laminin, and presumably proteoglycans. PYS-2 cells cultured on coverslips were extracted with 0.5% deoxycholate for 30 min at 0°C which resulted in removal of cells. Fixed coverslips were processed for SEM in JSM 35C with resolution of 70 2k. In low magnification, the matrix appeared as amorphous homogenous lamellar material. In higher magnifications, the lamellae were resolved into a meshwork of fine fibrillarity often embedded in amorphous material. Fibrils could be 10/zm long and fibril diameter in goldevaporated preparations measured to about 16 nm. Numerous spherical ca. 40-rim deposits were attached along the fibrils. Our previous immunofluorescence results indicate that type IV collagen, laminin, and proteoglycans are homogenously distributed throughout PYS-2 matrices. T h e observed ultrastructural features of PYS-2 matrix may reflect its molecular composition and that of corresponding early embryonic basement membranes known from immunofluorescence studies to contain the same components.

Altered Surface Activities and Locomotion of Monocytes in Hodgkin's Disease. A SEM and Time Lapse Study. HAAKON MELSOM, EINAR WIBE, TORE GODAL, AND ALBRECHT RHTI~, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital,

Montebello, Oslo 3, Norway. Cell clustering was found to develop in 24-hr cultures of monocytes from patients with untreated Hodgkin's disease (H.D.). Monocytes derived from normal blood donors did not show this phenomenon. The clustering depends on intact monocyte metabolism since incubation at 4°C abolished the reaction. By SEM H.D. monocytes showed an increased surface activity characterised by numerous large thin foldings covering the total cell surface. Time lapse phase contrast microcinematography revealed a strongly altered H.D. monocyte locomotion. H.D. monocytes from three different patients were analysed and locomotion of 10 cells per case measured. Using a semiautomatic image analysis the total distance covered was estimated and the distance between the sta~:fing point and endpoint was measured. The H.D. monocyte locomotion was 25.6 2 25.4 (range 4.1-53.6)/xm/hr, compared to 89.2 _+ 15.5 (range 75.0-105.7)/xm/hr for normal monocytes. The distance between starting points and endpoints was only one-fifth compared to normal monocytes. The study demonstrates the importance of time lapse cinematographic studies in analysing SEM surface characteristics such as fold-like membrane ruffles.

Transformation, Progression, and Differentiation of Ovarian Epithelial Surface Neoplasms: An Ultrastructural Study. F. STENB~CK AND R. VXXN~NEN, Department of Pa-

thology, University of Kuopio, SF-70101 Kuopio 10, and Department of Pathology, University of Oulu, SF-90220 Oulu 22, Finland. In this study the ultrastructural characteristics of ovarian epithelial surface tumors of mucinous, serous, endometrioid, and mesonephroid type and varying degrees of malignancy, benign, borderline, and malignant were studied and related to normal surface

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epithelium and histological findings in order to relate biological behavior to morphological characteristics. For this purpose 50 tumors were analyzed for occurrence of different types of cells and their ultrastructural characteristics were studied by transmission (TEM) and scanning electron microscopy (SEM). Histological analysis was also performed using conventional histological and histochemical methods. The results showed individual variations within the same neoplasm differences between tumors of the same histological type, as well as similarities between different types of tumors. Mucinous cystadenomas contained ciliated and microvillous epithelial cells with intestinal endocervical features; serous cystadenomas also contained cells with endometrial and tubal features. Endometrioid-type tumors contained mainly microvillous cells seen also in mesonephroid tumors. In tumors with a high degree of anaplasia the amount and development of surface structures decreased. The findings were verified by light microscopy. Morphological examination of the different types of tumors revealed the occurrence of ciliated and nonciliated columnar cells and goblet cells in all types though varying in frequency, with argentaffin cells also seen in some mucinous cystadenomas. It is concluded that ovarian epithelial tumors have a coelomic origin evolving through a metaplastic process in different directions. Ultrastructural analysis is useful in differentiating between different types of tumors, particularly highly malignant ones and also in assessing mode of progression.

Ultrastructural Identification of Virus in Oral Papillomas. Lis ANDERSEN AND OLE FEJERSKOV, Departments of Oral Pathology and Dental Pathology and Operative Den-

tistry, Royal Dental College, Aarhus, Denmark. The oral papilloma is a common benign neoplasm originating from the surface epithelium. It is analogous to the skin wart--verruca vulgaris. Although it has been established that in at least a considerable percentage of cases the skin wart is caused by virus, this has never been shown to be true of the human oral papilloma. Tissue from eight cases of oral papillomas was examined. The epithelium showed an acantomatous hyperplasia arranged in a papillomatous pattern. There was a conspicuous hyperkeratinization, predominantly hyperorthokeratinization. In the grooves between the papillomatous elevations a distinct stratum granulosum might occur, often exhibiting very large keratohyalin granules. The basic differentiation pattern of the keratinizing, squamous epithelium was normal. This pattern was cytologically reflected by the following structural changes from basal toward surface layers: involution of metabolically active organelles, increment of tonofilaments coupled with the appearance of membrane-coating granules, keratohyalin granules, and a keratin pattern. The nuclei of cells in the stratum spinosum and the stratum granulosum frequently exhibited very large nucleoli with aggregations of the nucleonema and vacuolization of the pars amorpha. In addition, accumulations of intranuclear filaments were observed. Virus particles were identified, but only in nuclei of granular and parakeratinized cell layers. The identification of virus particles and the changed morphology of nuclei in concert suggest a viral etiology of human oral papillomas.

Elemental Analysis of Histopathologically Defined Oral Carcinoma in Men. JOANNA WR()BLEWSKI,* MATTI ANNIKO,? AND ROMUALD WR6BLEWSKI,:~*Dental Faculty, Ka-

rolinska Institute, ?Department of Otolaryngology, Karolinska Sjukhuset, and SWennerGrens Institute, University of Stockholm, Stockholm, Sweden. X-Ray microanalysis was performed on 16-~m-thick cryosections of specimens from

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two types of oral carcinoma. To enable orientation and cell-type determination adjacent cryosections were stained with hematoxylin-eosin and examined in a light microscope. This procedure permits determination and analysis of neoplastically transformed cells and nontumor cells with regard to their elemental composition. The first tumor, prior to treatment, histopathologically classified as a low-differentiated squamous cell carcinoma, had been irradiated with 50 Gy. Macroscopically a complete remission had occurred after irradiation. Elemental analysis revealed elevated concentrations of sodium and chlorine and decreased concentration of potassium. The second tumor was a low-differentiated mucoepidermoid carcinoma located below an intact layer of mucous membrane. The elemental composition showed increased levels of sodium, phosphorus, and chlorine. Also potassium concentration was increased. The X-ray microanalytical technique in combination with light microscopy indicates an abnormal elemental composition extended into histopathologically normal tissue. A completely different elemental composition was found between irradiated and nonirradiated carcinoma.

Ultrastructural Features of the Mineralization Pattern in an Ameloblastic Fibroodontoma.

KAJ JOSEPHSEN, AKE LARSSON,* AND OLE FEJERSKOV, Departments of Anatomy, Dental

Pathology and Operative Dentistry, Electron Microscopy, and Oral Pathology, The Royal Dental College, Aarhus, Denmark, and *Department of Oral Pathology, Faculty of Dentistry, University of Lund, Malm6, Sweden. An ameloblastic fibroodontoma is a benign neoplasm composed of proliferating odontogenic epithelium embedded in a cellular mesodermal tissue. The tumor contains varying amounts of dentin and enamel-like tissue. Part of the epithelial tumor tissue appeared as isolated islands containing calcified material. The cell layers forming the wall of the epithelial islands had a great resemblance to those of the enamel organ during enamel formation. In the center of the islands areas of enamel-like tissue were mixed with vast areas of organic matrix which revealed two distinct components consisting of a network of tubular fibers and a fine granular homogeneous matrix, respectively. The tubular network was always found in relation to the epithelial cells and could in some areas be followed directly to the growing front of the enamel-like tissue. More often, however, the spacings between the tubular fibers at a certain distance from the cells became gradually obliterated by the fine-granular matrix component that further centrally coalesced to a coherent area in which only a few tubular elements were recognized. The fine-granular matrix was seen to adjoin enamel-like crystals and to separate large prism-like structures. The tubular fibers were haphazardly orientated and had in cross section a diameter of about 150 A. Long crystals were often seen in close relationship with either longitudinally or cross-sectioned tubular fibers. Apart from the prism-like structures the calcified material included a limited number of well-demarcated dense spherical masses having a core of closely packed fine-granular crystals which was surrounded by a rim of needle-like crystals. The spherical masses were found as isolated islands in the fine-granular organic matrix. From the localization of the two matrix components in relation to the cells and the mineral components it is assumed that the tubular fibers are secretory products of the epithelial cells. A time-related breakdown of the tubular matrix component, eventually enzymatically, may explain a gradual replacement of the tubular fibers with the finegranular matrix component. It is further assumed that the tubular matrix component directs the formation of long enamel-like crystals while the fine-granular matrix, most likely consisting of degraded tubular protein, has lost this property. The latter, however, may mediate the formation of calcified dense spherical masses. Similar ultrastructural

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mineralization patterns have not previously been described in normal mineralizing tissue of epithelial or mesenchymal origin.

The Distribution and Ultrastructure of Subgingival Plaque in Beagle Dogs with Gingival Inflammation. JORGEN THEILADE AND ROLF ATTSTROM, Departments of Electron Mi-

croscopy and Periodontology, Royal Dental College, Aarhus, Denmark. The present study was designed to describe the distribution and ultrastructure of subgingival plaque in beagle dogs in an attempt to establish a correlation between these deposits and the adjacent soft tissue destruction. Biopsies comprising one premolar with adjacent buccal gingiva were obtained from five approximately 2-year-old beagle dogs in which chronic gingivitis was diagnosed clinically. After prefixation of the biopsies and decalcification of the teeth, the gingival biopsies were cut axially to yield 0.5-mm-thick slices, after which the tissue was postfixed and embedded in Epon. From each dog one slice was randomly selected for the study. Each block was semiserially sectioned to provide axial sections. From the blocks one semithin section for light microscopy and an adjacent thin section were cut for every 50/xm in mesiodistal direction. Light microscopy revealed that in three of the biopsies examined no plaque colonies were present on the tooth. In two biopsies, varying numbers of subgingival plaque colonies were present. Electron microscopy revealed that in one of the biopsies Gram-negative cocci or rods predominated all subgingival colonies observed. In the other biopsy in which subgingival colonies were present spirochetes predominated. In all five biopsies single or small groups of microorganisms were observed either between the tooth and the junctional epithelium or between the most superficial cells of the latter. Evidence of phagocytosis of microorganisms was seen, but to a limited extent. The distance from the gingival margin to the most apical subgingival plaque averaged 0.25 mm (SD _+0.05 ram). The distance from the gingivat margin to the most coronal hemidesmosome was 0.43 mm (SD _+0.06 ram) and this distance did not seem to be influenced by the presence or absence of subgingival plaque colonies in the section or in the biopsy slice. The results indicate that subgingival growth of bacteria on the tooth surface is not necessarily responsible for the destruction of the subjacent junctional epithelium.

Differentiation of Autologous Skin Grafts in the Oral Cavity. LARS NYBROE AND LIS

ANDERSEN, Departments of Oral Surgery and Oral Pathology, Royal Dental College, Aarhus, Denmark. Skin grafts are commonly used in preprosthetic surgery in the oral cavity with the purpose of establishing a proper buccal sulcus in patients with marked atrophy of the alveolar process. The aim was to evaluate the long-term effect of the oral environment on split skin grafts, with special emphasis on epithelial differentiation. The material comprised biopsies from 17 individuals having received skin grafts 10 years previously. The grafted skin retained the structural characteristics for skin. However, changes in epithelial histology and keratinocyte differentiation were encountered. Within each biopsy the grafted skin showed regions with structural variations of the following two patterns: epithelial hyperplasia, accumulations of glycogen, altered keratinization, and inflammation in the connective tissue or normal to slight atrophic epithelium, hyperpigmentation, normal keratinization, and absence of inflammation subepithelially. The maintenance of epithelial specificity in split skin grafted to the oral cavity 10 years preciously supports the concept of a dominating influence from the dermal compartment contained within the graft. The

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changes in epithelial differentiation pattern seem closely related to presence or absence of subepithelial inflammation caused by pressure from the dentures and/or plaque on the fitting surface of the dentures.

Early Microbial Colonization of Human Tooth Surfaces: A SEM Study. B. NYVAD AND O. FEJERSKOV, Department of Dental Pathology and Operative Dentistry, Royal Dental

College, Aarhus, and Institute of Anatomy, University of Odense, Denmark. So far most electron microscopical studies of early microbial colonization of teeth have been carried out on different artificial surfaces (e.g., plastic films, Mylar strips, combined Epon and hydroxyapatite preparations). However, biochemical properties of such surfaces have been demonstrated to differ significantly from that of natural tooth surfaces. Furthermore, the microstructure of tooth surfaces may influence the pattern of early microbial colonization. The aim of the present investigation was therefore to study early microbial colonization on human dental enamel and cementum in vivo by SEM. Specimens were cut from unerupted third molars and exposed to the oral environment for periods of 4 to 48 hr. After ultrasonic treatment the test pieces were mounted in an intraoral appliance which was inserted along the lower buccal segments. Immediately after removal the tooth surfaces were fixed in a combined paraformaldehyde/glutaraldehyde fixative for 4 hr, and postfixed for 2 hr in 2% osmium tetroxide. The specimens were dehydrated, critical point dried, and gold coated prior to SEM examination. Surprisingly few microorganisms adhered to both cementum and enamel 4 hr after exposure, but the surfaces were entirely covered by a fine granular coating, possibly of proteinaceous nature. On the enamel cocci colonized initially in pits and surface irregularities. After 8 hr scattered cocci had spread as a monolayer along the perichymata of the enamel surface, whereas a profuse haphazard distribution was observed on the cementum. Concurrently, a multilayered growth was seen in the center of the microcolonies. Twelve- to twentyfour-hour specimens revealed a thick homogeneous coccoid layer covering all the surfaces. During the following 24 hr a further thickening of microbial deposits was observed with single rods and filaments extending perpendicular to the surface. In conclusion: (1) Early dental plaque formation can be characterized by an initial lag phase of a few hours during which a limited number of microorganisms adhere. This phase is followed by a rapid growth and proliferation. (2) The pattern of colonization is determined by the surface structure of the tooth. (3) Dental cementum appears to become colonized more rapidly than dental enamel.

Electron Microscopic Studies on Precipitated Calcium Phosphate in Denitrifying Bacterial Films. G. HOLM KRISTENSEN,* F. CHRISTENSEN,t AND E. ARVIN,* *Department of San-

itary Engineering, Technical University of Denmark, Building 115, and tDepartment of Biochemistry and Nutrition, Technical University of Denmark, Building 224, DK 2800 Lyngby, Denmark. At the Department of Sanitary Engineering reaction kinetics of bacterial films (biofilms) has been investigated for several years. One project deals with precipitation of calcium phosphate inside denitrifying biofilms. The precipitation is a consequence of the high pH value created in the biofilm by a combination of the production of alkalinity and the diffusional resistance to out-transport of alkalinity. Results from chemical and X-ray spectrum analyses indicate the precipitate to be hydroxyapatite, possibly with carbonate substitution. Interference contrast microscopy shows that precipitation is concentrated in the deeper part of the biofilm. TEM reveals that the precipitation generally is initiated

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just outside the bacterial membranes. This phenomenon is likely due to an alkaline microenvironment in the near surroundings of the bacteria. The formed crystals are needle shaped, length around 500-1000 ]k, length to thickness ratio av 20:1. The outline of cavities in the aggregated precipitate indicates that the bacteria die from being " f e n c e d " into solids. Some of the bacteria apparently have the ability to duplicate themselves via hyphae growing out through the precipitate, thereby facilitating continuation of the biofilm formation.

A Semicrystalline Body in Cells ofAcholeplasma laidlawii. C. WEIBULL,T. CLEMENTZ, AND L. RILFORS, Department of Microbiology, University of Lund, S-223 62 Lund, Swe-

den. Acholeplasma laidlawii A, strain EF22, was grown in a lipid-depleted tryptose/bovine serum albumin medium at 37°C. The medium was supplemented with palmitic and oleic acid. The cells were harvested in the late exponential growth phase, washed with a Tris buffer, and fixed with 1% glutaraldehyde for 30 min at 0°C. After postfixation with OsO4 for 1 hr at 0°C the cells were dehydrated with ethanol and embedded in Epon. Sections of the cured embeddings were stained with UO.,Ac. In many cells dark, elongated bodies having a hexagonal cross-section were seen. Some of the bodies exhibited a regular striation with a repeat distance of approximately 5 nm. Similar bodies were seen in cells fixed only with glutaraldehyde. To our knowledge the occurrence of bodies in A. laidlawii of the appearance just described has not been reported elsewhere. They may represent a nonlamellar lipid phase or be of protein nature, possibly an actin-like substance.

Electron Microscopical Investigation of Soil Bacteria: Comparison of Cells Released from Soil and Cells Isolated as Pure Cultures. L. BAKKEY, O. A. OLSEN, AND T. KREKLING,*

Microbiological Institute and *Service Institute, Agricultural University of Norway, 1432 As-NLH, Norway. Studies have shown that the number of structurally intact cells in soil exceeds the number of cells forming colonies on conventional agar media with a factor of 102-103. We have reduced this to a factor of 10, by introducing new media with filter-sterilized soil extracts and an extremely low content of nutrients (carbon). By using TEM and SEM, we have compared the morphology of bacterial cells released from soil with organisms isolated as pure cultures. There is a remarkable diversity in the morphology of the cells released from soil. Among these, we have been able to isolate and keep in pure cultures budding bacteria and irregularly formed rods and cocci. In addition, soil contained a large number of very small cells (0 < 0.5/zm) as well as cells surrounded by an extracellular substance, which we up to now have not been able to isolate as pure cultures. However, by modifying our new media, it should be possible to isolate and characterize most of the bacterial flora common in soil. In accordance with earlier findings, bacteria with unusual forms, as well as cells with extracellular material were frequently seen in TEM preparations from soil. Furthermore, from pure cultures of ordinary-shaped cells, we occasionally observed amorphous cell-like structures, apparently surrounded by membranes, and cells of unusual shapes. Comparative studies by using SEM indicated that at least some of these unusual forms may be due to artifacts and/or unfavorable orientations of the section plane. Such findings could easily be erroneously interpreted as new structural forms. We strongly recommend that material prepared for TEM should also be investigated in SEM.

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Grain Development in Normal and High-Lysine Barley. O.-A. OLSEN* AND T. KREK-

LING,t *Institute of Genetics and Plant Breeding, and ~fService Institute, Agricultural University of Norway, 1432 fiS-NLH, Norway. During the period from pollination to Day 33, seeds from the Ris~b high-lysine mutants Nos. 58, 86, and 1508 were compared with their respective motherlines by means of light and electron microscopy, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants showed reduced seed growth rates, a fact due to lower dry matter content per cell. The number of endosperm cells was the same in all lines. In mutants 56 and 1508 cell growth stopped after 21 days, whereas in mutant 86 and the motherlines cell size doubled between Days 15 and 33. A reduced starch deposition could account for the lighter grains of the mutants. In mutants 56 and 1508, half of the reduction was due to an 80% reduction in the number of small starch granules. In mutant 86, only a reduction in the number of large starch granules was found. Mutant 56 had a reduced amount of Bhordeins, whereas in mutant 1508, B- and C-hordeins were almost absent. In mutant 86, a delayed onset of the hordein synthesis was revealed. There was a good correspondence between the amount of hordein present and the total content of protein bodies per cell. The altered protein body morphology in the mutants could be explained by assuming the presence of prolamins in two of the protein body components described.

The Ultrastructure of the Cryptophycean Flagellate Hemiselmis anomala Butcher, with Special Emphasis on the Flagellar Apparatus. KIM PEITER JORGENSEN, Institut for Spo-

replanter, KCbenhavns Universitet, Ostre Farimagsgade 2 D,D.K. 1353, Denmark. The genus Hemiselmis has general biological interest for two reasons: (1) This minute flagellate is sometimes a dominant nannoplankton algae in seawater, i.e., it is ecologically important; (2) the class possesses a number of primitive characters and is therefore interesting anatomically. In the light of the about 20 published papers about the class, this investigation seems to indicate that this particular genus is the most unspecialised so far seen in this class, and it is therefore perhaps the most unspecialised flagellate yet sectioned. H. anomala has unusually elaborated flagellar hairs. The ways these hairs seem to travel to their position on the flagella will be mentioned. The flagellar root system is weakly developed, but is still the main strengthening system in the cell. It is important in view of the stress put on a wall-less cell travelling 30-50 times its own length per second.