3D reconstructions of organelles and their fine structures

Research project P 13614 3D reconstructions of organelles and their fine struktures Günther ZELLNIG 28.06.1999 Transmission electron microscopy (TEM) is usually used for the 2D investigation of the ultrastructure of cells and their organelles (SITTE 1991, SMITH & WOOD 1996). However, it is not possible to specify the relations in the cell and organelle because ultrathin sections of plant tissue are about 80 mm thin for evaluation in the TEM. Therefore, only a very small part of the cell or organelle is visible and calculations of areas or volumes of organelles and fine structures are only speculative or provide uncertain data. We use a method similar to that described by OSUMI 1998 to get 2D data and 313 images of different organelles based on TEM serial sections and computer reconstructions (ZELLNIG et al. 1996, PERKTOLD et al. 1998). The total reconstruction of cell units generally gives an insight, how closely organelles can be packed and how subcellular structures are arranged. As an additional innovation, our method allows to show distinct structures of interest, and other systems (organelles or their substructures) can be faded out. Besides, selected structures can be viewed from various sides. In this manner it is possible to obtain more detailed information about the association of organelles or structural continuities. Distinct areas of selected organelles, like the thylakoid surface, stroma, starch and plastoglobuli of plastids, can be measured by a digital imaging system. The volume of these structures is calculated by the thickness of the sections. Cell organelles and their ultrastructure are affected by different stress factors (FINK 1988, BÄCK et aL. 1994), but mainly plastids were investigated because alterations/damages were noted mainly in these organelles. However, the evaluation of the condition of this organelle based on the information of a limited number of sections. Moreover, other organelles like mitochondria, or peroxisomes are more difficult to examine due to their small average size and changes in their structures and areas or volumes can hardly be noticed with conventional methods. Therefore, 3D reconstructions and area/volume measurements are of great importance and urgently needed for an exact registration of structural relationships. An exact distinction between damaged or daily periodic variable organelles is only possible with these informations. We intend to investigate the 3D fine structure of various cell organelles; during diurnal rhythm of selected plants (Picea abies, Pinus canariensis and Spinacia oleracea) under defined conditions in climate chambers. Additionally, the influence of drought will be registered. The result of all of these investigations is a more detailed knowledge about the architecture and the distribution/variation of fine structures, including areas and volumes on a very high level of resolution. It is important to stress that this kind of investigations lead to reconstructed, real existing cell structures and are not models based on statistical possibilities. These fundamental data will be essential for both further classifications of structural parameters and the interpretation of the functional significance of physiological and biochemical data.

Chloroplasts of three species spinach, spruce and pine are the main object of investigation in this project. These are the organelles in green plant cells, which are responsible for the photo-synthesis, a process where light energy from the sun is converted into chemical energy and used to produce carbohydrates. The fine structures of chloroplasts can only be seen in the transmis-sion electron microscope (TEM), therefore the organelles have to be cut in very thin sections of a ten thousandth part of a millimeter. After investigation with the TEM, 3D reconstructions are possible by digital imaging programs. These procedures allow for the first time the 3D recon-struction of complete chloroplasts and the quantification of their fine structures. By means of these investigations daily periodic variations of fine structures can be distinguished from changes caused e.g. by drought stress. Our results clearly show differences in the chloroplast structures during the day and after drought stress, which are variable in the investigated species. This knowledge has to be regarded as fundamental for further investigations. With conventional electron microscopical methods it is not possible to specify the relations of fine structures in the cell and organelles because ultrathin sections of plant tissue are about 80-100 nm thin for evaluation in the TEM. Organelles like chloroplasts have a size of about 4-8 m, therefore only a very small part of the cell or organelle is visible and calculations of areas or volumes of organelles and their fine structures are only speculative or provide uncertain data. Cell organelles and their ultrastructure are affected by different stress factors, but mainly plas-tids were investigated because alterations or damages were noted mainly in these organelles. However, the evaluation of the condition of this organelle is based on the information of a lim-ited number of sections. Moreover, other organelles like mitochondria or peroxisomes are more difficult to examine due to their small size of about 0.5-2 m in average and changes in their structures and areas or volumes can hardly be noticed with conventional methods. Therefore, 3D reconstructions and area/volume measurements are of great importance and urgently needed for an exact registration of structural relationships. An exact distinction between damaged or daily periodic variable organelles is only possible with the data obtained from our investiga-tions. With our method 2D data and 3D images of different organelles based on ultrathin serial sec-tions and computer reconstructions can be obtained. The total reconstruction of cell units gen-erally gives an insight on how closely organelles can be packed and how subcellular structures are arranged. As an additional innovation it is possible to show distinct structures of interest, and other systems (organelles or their ultrastructures) can be faded out. Besides that, selected structures can be viewed from various sides. In this manner it is possible to obtain more de-tailed information about the association of organelles and their structural continuities. Distinct areas of selected organelles, like the thylakoid surface or starch grains in plastids, can be meas-ured by a digital imaging system. It is important to stress that this kind of investigations lead to reconstructed, real existing cell structures and are not just models based on statistical possibili-ties. The result of all these investigations is a more detailed knowledge about the architecture and the distribution or variation of fine structures, including areas and volumes on a very high level of resolution. These fundamental data are essential for both further classifications of structural parameters and the interpretation of the functional significance of physiological and biochemi-cal data.