The "Structural Cell Biology Group" as the core facility for light and electron microscopy at the Institute fulfils a dual function: (a) it provides practical and theoretical advise to other groups that wish to address research problems by sophisticated techniques of light and electron microscopy and (b) conducting own research projects. Among ultrastructural analysis and 3-D reconstructions we mainly focus our interests on the spatial distribution of plant enzymes and molecules as analyzed by immunogold electron microscopy or fluorescence microscopy. Owing to the state-of-the-art technology in light and electron microscopy, the research group has established and offers several routine and advanced methods in microscopy and sample preparation that enable guest researchers to address a large variety of ultrastructural problems at the levels of tissue, cell, compartment and molecule.
The present technology platform covers the following instruments and provides therefore an excellent technical platform for modern cell biology: 

- Transmission electron microscope FEI Tecnai G²-Sphera 200 KV

- Field emission scanning electron microscope FESEM Hitachi S 4100

- Confocal laser scanning microscope Zeiss LSM 510 META

- Fluorescence microscope Zeiss Axioskop + Zeiss AxioCam HRc camera system

- Fluorescence microscope Zeiss Axiovert 135 + Zeiss AxioCam HRc camera system 

Confocal Laser Scanning Microscopy (CLSM) is a valuable tool for obtaining high resolution images and 3-D reconstruction at the resolution of the light microscope. The key feature is its ability to produce blur free images by thin optical sectioning. The use of 3-D reconstruction of a Z-series enables the visualisation of larger structures or compartments. Genetechnology and a great variety of specific fluorescence antibodies and dyes offer CLSM a wide field of application. One of the major features in plant research is the monitoring of cell dynamic processes and the localisation of fluorescent labelled molecules in living cells and tissues by the use of sophisticated techniques (fusion proteins, particle bombardment, fluorescence labelling and staining) to answer open questions about plant morphology or the mechanism of primary and secondary metabolic pathways. To visualize distance dependent protein-protein interactions Fluorescence Resonance Energy Transfer (FRET) or “tools“ like Cameleon and “splitting GFP“ are used. Plant tissue is characterized by various autofluorescence signals (chlorophyll, lignin). The overlapping emission spectra with a number of widely used fluorescent dyes, causes very often problems for the signal detection. With Emission Fingerprinting of the Zeiss LSM 510 META, the highly efficient optical grating onto 32 channels of the META detector, allows the separation of complex fluorescence signals - even with widely overlapping emission spectra - is an easy, three-step task. Additional technical features as multitracking, lamda-scan or Acousto-Optical Tunable Filter (AOTF) improve the signal detection and therefore cell viability of examined tissue. Finally this increases the efficiency of optical sectioning, detection of weak fluorescence signals, monitoring of cell dynamic processes by using single – or time laps imaging or cell biological methods like FRET or FRAP (Fluorescence Recovery after Photobleaching). Routine examinations of fluorescence microscopy and histology are supported two light microscopes Zeiss Axioskop and Zeiss Axiovert 135, each provided with a CCD-Camera system Zeiss AxioCam. 

The acquisition of a Laser Scanning Microscope with a dual scan head (combination of “line scanning“ and point scanning with a 2-photon laser) in 2007/2008 would be an important enhancement of the technical imaging platform. The intrinsic property of a 2-photon laser is that they give off photons in pulses in less than 10 ns intervals. The use of longer wave length is inherently less damaging to biological material than shorter wave lengths of CLSM and is penetrating approximately 5 times deeper into biological material. With up to 120 confocal images, the “line scanner” films up to twenty times more images per second than any conventional confocal system with significant improved sensitivity. The ultra-fast CCD line detector allows parallel imaging of 512 pixels with high quantum yield. Therefore the dual scan system would significant improve notably the monitoring of fast cellular processes, Z-stack imaging, life cell and time laps imaging.

TEM is most frequently used to examine the architecture of plant cells and localization of macromolecules, particularly proteins, in cell compartments. Conventional chemical fixation (aldehyde, osmium) is used for routine ultrastructural research (Lowicryl, Spurr, LR White). Steadily growing is the interest in the cellular distribution and localization of proteins and enzymes in wild type and transgenic plants employing TEM. Currently the most advanced method to address these problems is use of immunogold electron microscopy. To ensure that the cell structure and momentum location of a particular protein do not change, workable techniques of sample cryo-fixation and cryo-substitution for plant and animal tissues and micro-organisms with a relative high through-put have been established. Even High-Pressure-Freezing (HPF) can be applied for special research purposes and is being improved technically for other applications. However, to date the most frequently used technique of low-destructive sample preparation giving rise to high protein immunolabelling is Progressive Lowering of Temperature (PTL). Our new 200 kV TEM with a Gatan Imaging filter GIF2002 and a motorized goniometer offers us the application of 3D-electron tomography and Electron Energy Loss Spectroscopy (EELS). Cellular tomography using transmission electron microscopy (TEM) is the only available technology to chart the inside of a cell. Imaging the cellular structures in all three dimensions to generate an accurate three-dimensional map of the interior of the cell, enables researchers to analyze molecular structures in relation to the cellular architecture, the cytoskeleton and the cell organelles. Most proteins do not function as individual entities, but in coordination or dependence with other proteins. The knowledge of the three-dimensional organization is essential to understand protein function at the cellular level. Digital imaging is carried out with a SIS MegaView III (side mount) and a Gatan 2k x 2k CCD-Camera (bottom mount). The upgrading of the TEM for cryoelectron microscopy of is planned. 

The Hitachi S 4100 Field emission scanning electron microscope is equipped with a backscatterdetector and cryotransfer chamber (Oxford CT 1500 C) thus enabling a wide range of applications. The SEM  is mainly used for surface studies. Next to classic studies of taxonimic characters and of morphological alterations in transgenic plants, the field-emission-gun technology also allows for high resolution studies well into the nano range. With the help of the cryo-unit it is also possible to study frozen samples. Deep-freezing of plant tissues is a quick and easy procedure and is less likely to produce artefacts compared to the critical-point drying method. Not only can surface structures be studied but frozen sections can also be cleft enabling investigations of cell interiors. The cryo-application is also very useful in assessing the effectiveness of cryo-preservation protocols for explants. In this procedure cryo-preserved explants are cleft and the extent of ice crystal formation within the interior of the cell can be used an indicator of the extend of cryo-induced cell damages. The backscatter detector, enabling the detection of primary electrons, is a valuable tool for analyzing immunolabelling experiments. In these experiments tissues or cell fractions are labelled with gold-conjugated antibodies, often in combination with silver- or gold-enhancement. Whereas the soft parts are visualized with the normal, secondary electron, detector, the heavy metal components of the immunolabel can be selectively visualized with the backscatter detector.

The concentration of TEM, SEM, LM and CLSM within one research facility enables the use of various cell biological methods to confirm experimental results.

Ongoing internal and external cooperative projects:

1. Comparative morphology and ultrastructure of the storage parenchyma in seeds of Pisum sativum WT and AGP knock-out mutant (Collaboration with the Research Group Plant Reproduction Biology).
2. Examination of the morphology of potato shoot-tips after cryoconservation (Collaboration with the Research Group Cryopreservation).
3. Comparative ultra structural characterisation of leaf of wild type and transgenic plants of Solanum tuberousum expressing bacterial flavodoxin from Anabaena growing under various stress conditions (Research Group Molecular Plant Physiology).
4. Investigation of the ultrastructure of kinetochores in Luzula elegans (Collaboration with the Research Group Chromosome Function and Structure).
5. Structural investigation of seed degeneration in shoots of the Arabidopsis thaliana knock-out mutant Aurora (Collaboration with the Research Group Chromosome Function and Structure).
6. Immunological localization studies of the Jekyll protein in seeds of Triticum aestivum (Collaboration with the Research Group Gene Expression).
7. Ultrastructural study of plastids in the cotyledons of Vicia napus (Collaboration with the Research Group Gene Regulation).
8. Ultrastructural characterisation of homozygous topoisomerase 3A mutants of Arabidopsis thaliana  (Institute of Botany II, University of Karlsruhe, Karlsruhe, Germany).
9. Comparative ultrastructural characterisation of rosette leaf tissue of Arabidopsis thaliana Col0, various mutants of the Digalactosyldiacyglycerol Synthase genes and DGD1 over-expressing lines (Collaboration with the Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).
10. Comparative ultrastructural and immunocytochemical characterisation of flower tissue of Arabidopsis thaliana Col0 and various Phosphaditylserine Decarboxylase mutants (Collaboration with the Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).
11. Comparative ultrastructural and immunocytochemical characterisation of root and leaf tissue of Arabidopsis thaliana Col0, ANTR-5 mutants and over-expressing lines (Collaboration with the Institute Plant Physiology, University of Kaiserslautern, Germany).
12. Ultrastructural characterisation of hipI-SOD antisense plants of Populus tremula ssp. tremuloides and localisation of hipI-SOD (Collaboration with the Dept. of Forest Genetics and Plant Physiology, Swedish University of Agricultural Science Umeå, Sweden).
13. Comparative ultrastructural characterisation of oxidative stress mutants of Arabidopsis  thaliana (Collaboration with the Dept. of Plant Physiology, University of Stockholm, Stockholm, Sweden).
14. Comparative morphological and ultrastructural characterisation of stem and siliques of wild type and transgenic Arabidopsis plants srrp1-1 and ssrp1-2 (Collaboration with the Dept. of Life Sciences, University of Aalborg, Aalborg, Danmark).
15. Examination of protein-protein interactions of the DNA recombination in rice (Collaboration with the Bhabha Atomic Research Center, Molecular Biology and Agriculture Division, Bombay, India).
16. Examination of the supramolecular organization of the synchronisation of light and dark reaction in the photosynthesis apparatus of the bacteria Synechococcus und Synechocystis (Collaboration with the Bhabha Atomic Research Center, Molecular Biology and Agriculture Division, Bombay, India).