Abstracts of The Netherlands Society of Electron Microscopy 457

ARE THE STOMATA OF VICIA FABA L. CLOSED IN THE DARK?

J. Aben, F. Thiel* and A. Boekestein*

KE,YA, Dept. Environmental Research, Amheim; *Techn. and Phys. Eng. Res. Service,

Waqeninqen, The Netherlands

Air pollution may affect growth and production of agricultural crops, and the role of the stomata is in this respect crucial because most of the pol- lutants enter the plant system through these adjustable openings in the leaf surface. Stomata1 diffusion resistance is normally derived from the transpira- tion rate. However, this is only justi- fied if transpiration is entirely under stomata1 control. Because Vicia faba shows dark transoir? ‘tion rates uu to 40% of rates observed at light saturation conditions, it is questioned whether the stomata of Vicia faba are really closed in the dark. Samples of upper and lower sides of light and dark adapted leaves were taken and cryofixed by immersion in nitrogen slush. After gold sputtering, specimens were observed in the Philips SEM535 equipped with the Hexland CT1000 cryostage at 20 kV accelerating voltage. It appeared that stomata on top of the leaves were closed in the dark while stomata on the lower side of the leaves were still open in the dark. However, these stomata were not completely opened when compared to the situation at light saturation. It also appeared that the number of stomata on top of the leaves was less than at the lower side and that they were also less opened at light saturation.

ULTRASTRUCTURAL LOCALISATION OF ACID PHOSPHATASE IN HUMAN-BLOOD-DERIVED DENDRITIC CELLS AND MONOCYTES

J.M.S. Arkema, P.A.J.M. de Laat, M.A.M. Verdaasdonk and E.C.M. Hoefsmit

Department of Cell Biology, Medical Faculty, Free University, P.O. Box 716>1, NL-1007 MC Amsterdam, The Netherlands

On the light microscopical level, besides other characteristics, dendritic cells (DC) can be discriminated from cells of the monocyte/macrophage (ma/m@) lineage by the presence of acid phospha- tase (AcP) activity in a spot near the nucleus, whereas ma/m@ show AcP activity throughout the cytoplasm.

The aim of our study was to investi- gate the exact subcellular distribution of AcP in both cell types enriched from human peripheral blood. On non-adherent low-density cells (NALDC), containing DC and mo, enzyme cytochemistry for AcP was performed for light microscopy and elec- tron microscopy. All ceils showed little AcP activity but the localisation dif- fered. About 30% of the cells showed it in some lysosomal structures localized predominantly in an area near the nu- cleus. In other cells, AcP activity was observed in lysosomal structures local- ized throughout the cytoplasm. Further purification of DC, as established by an increased response in an allogeneic mixed lymphocte reaction, led to an in- crease of cells with AcP activity in a spot near the nucleus. We may conclude that in contrast to mo/mQ, DC show AcP positive lysosomes in a juxtanuclear position.

ELECTRON MICROSCOPY OF NUCLEIC ACIDS AND PROTEIN-NUCLEIC ACID COMPLEXES

A.C. Arnberg

Biochemical Laboratory, RUG, Groninqen, The Netherlands

The contribution of nucleic acid el- ectron microscopy in the developments in molecular biology has been considerable. Visualization in the electron microscope of double- and single-stranded nucleic acids, DNA and RNA, is possible using various modifications of the protein monolayer spreading technique. In these spreadings the nucleic acids appear ex- tended in a two-dimensional protein film. With this method information about the structural and functional organization of genetic material can be gained. The rapid advance of recombinant DNA cloning technology made it possible to isolate and study the structure of complex euca- ryotic split genes.

A more recent application of the spreading technique has been the charac- terization of products formed in proces- sing of precursor RNA transcripts. These products often show abnormal electro- phoretic mobility due to topological con- straint and their structure is not easily amenable to biochemical analysis. By electron microscopy some new topological forms of RNA were observed. Examples are branched circular RNAs (lariats) and in- terlocked RNA circles (catenanes).

The reproducibility and the resolution

of the spreading method make it possible to map specific regions with a precision of about 50 nucleotides with a minimum of material. Special methods have been developed for the study of protein DNA interactions. A direct visualizationof comple::es is possible but also the anal- ysis of protein-induced structural changes in DNA such as bending and looping. Some useful applications of nucleic acid electron microscopy are shown and some drawbacks discussed.

SCAlJNING TUNNELING MICROSCOPY OF COMPOUNDS WITH A LAYER-TYPE STRUCTURE

G.P.E.W. van Bakel, P.M. Bronsveld and J.Th.M. de Hosson

The principal aim of this research project is the investigation of the atom- ic structure of interfaces which are of great importance to materials science and technology. The main experimental tool explored is a scanning tunneling micro- scope. Scan sizes of scanning tunnelinq microscopes with atomic resolution are of the order of i urn. In order to locate interfaces it is necessary to be able to scan sample surfaces having mm dimensions. For this reason, a STM unit is currently being built inside a scanning electron microscope. UHV conditions are given special attention, for which purpose a sample chamber is under design, pumped by a Ti-sublimation unit.

In air, the surface of cleaved TiS2 is being investigated. The material was made by the laboratory of Inorganic Chemistry of the university. This has led to images having atomic resolution which revealed two different kinds of isolated defects with three-fold symmetry. The first con- sists of a triangle of 15 highlighted atoms, the second of a triangle of three highlighted atoms surrounded by three missing spots. These two phenomena occupy opposite crystallographic sites in the hexagonal surface lattice.

IMPROVED EPON EMBEDDING FOR BIOMATERIALS

E.H. Blaauw, J.A. Oosterbaan and J.M. Schakenraad

iicpdrtment ot Hlstoloijg Jnd CL 1 i i{1c,Loyq,

I:nivtjr.slty of Gron~nyi~n, (i~,osti,r.~;~!ijc,i i>'i/.?,

To obtain improved transmission elec- tron microscopy sections for cell biolo- gical and interface evaluation of im- planted biomaterials we present an im- proved embedding procedure. Standard problems in preparation and sectioning, like dissolution of the biomaterial, or holes and chatter in the sections, can be prevented by introducing butyl-2,3- epoxypropylether as an intermedium be- tween the dehydration series and the Epon resin. Most biomaterials were not affected by this chemical agent. The in- troduction of butyl-2,3_epoxypropyiether resulted in completely homoqeneous Epon blocks which enabled us to cut 50 nn sections, free of holes and chatter. The biomaterials did not dislodge during the process of sectioning, and the cell- polymer interface remained intact for electron microscopical evaluation.

AN ULTRA-HIGH-VACUUM CHAMBER FOR A PHILIPS EM430 STEM

A.J. Bleeker, F.J. Pijper and P. Kruit

At the Delft Particle Optics qroup i: new Scanning Transmission Microscop: based on the Philips F‘M43': 1s under de- sign. With this microsccpe it will be possible to do Auger spectroscopy. With the help of a parallelisinq field the Auger electrons will be guided from the sample to the energy-dispersive 180" electrostatic analyzer. Because of the combination of the high spatial resolu- tion of the STEM and the large accept- ance angle of the spectrometer this in- strument will be a powerful surface analytical instrument. However, to do Auger spectroscopy one needs to have a clean surface. This means the vacuum at the sample position must be in the Torr range or lower. To obtain thii"-lo vacuum the whole objective lens rcqion is redesigned: All elastomcre O-rings have been taken out, the pole pieces will be connected to the stainless steel vacuum chamber with a combined soldering and welding seal, the whole chamber will be sealed with Conflat seals and pumpinq will be done completely oil-free with an Iongetter pump and two Ti-sublimation pumps. UHV is not only important for surface analysis and material science,