[in Japanese]

View full abstract

Recent development of the 3D imaging has read us “to observe” the function as well as fine structure of cells and tissues. In this special issue, techniques and practice of four different methods for the 3D imaging: traditional serial ultra-thin sectioning, serial block-face scanning electron microscopy (SBF-SEM), focused ion beam scanning electron microscopy (FIB-SEM), X-ray micro-Computed Tomography and 2 photon microscope, are presented and discussed. This issue is based on the symposium held at the 78th Annual Meeting of the Botanical Society of Japan and co-hosted with The Japanese Society of Plant Morphology and Integrated Imaging Research Support.

View full abstract

Transmission electron microscopy (TEM) allows high-resolution imaging. However, it provides only two-dimensional information owing to the ultrathin sample sections of approximately 80 nm. To obtain three-dimensional (3D) images using TEM, additional methods are needed. There are two major techniques for 3D-TEM, computer tomography (CT) and ultrathin serial sectioning. Although the latter requires high technical skills and is time-consuming, it allows the 3D imaging of whole cells, even those that are relatively large in size, such as algal cells. We used the 3D-TEM technique to study Haematococcus pluvialis, a micro-green alga with relatively large cells, more than 30 µm in diameter. It was known that H. pluvialis accumulates strong antioxidant astaxanthin under stress conditions, but little information was available on the ultrastructural changes during the accumulation. We performed a 3D-TEM reconstruction based on over 370 serial sections per cell to visualize the dynamics of ultrastructural changes during the accumulation. This study showed that the oil droplets containing astaxanthin increased from 0.2% of relative volume in a green cell to 52% of relative volume in a red-cyst and that the chloroplast volume decreased from 42% to 10% during the accumulation. In this review, we present the results of this study and discuss the effectiveness and technical aspects of the 3D-TEM technique.

View full abstract

Serial block-face scanning electron microscopy (SBF-SEM) is an advanced 3-D electron microscopy for investigating structures of large volume specimens such as a whole cell or a piece of tissues at a resolution better than a few tens of nanometers. In this method, block surface of a resin embedded specimen is trimmed by a diamond knife attached to an in-chamber ultramicrotome, and the newly exposed surface is imaged by SEM. The sectioning and imaging can be repeated automatically to get serial block-face images of the specimen. The resultant 3-D structure is reconstructed from the serial block-face images after individual image alignment. This review shows the principle and performance of SBF-SEM, and introduces some recent applications using SBF-SEM. For example, morphological changes in mitochondria regulated by Cdc48p/p97 ATPase were recently examined by SBF-SEM. The Cdc48p is a highly conserved cytosolic AAA chaperone that involved in a wide range of cellular processes including the regulation of the mitochondrial morphology. The results demonstrate that SBF-SEM has considerable advantages in quantitative morphological analyses on organelles and intracellular structures in the whole cell.

View full abstract

The cellulolytic fungus Trichoderma reesei is a potent cellulase producer and, therefore, cellulase hyper-producing mutants have been developed. However, morphological feature has still remained to be analyzed for understanding phenotypic change of T. reesei mutants. In this review, we show an electron microscopic observation of T. reesei to obtain new insights of morphological phenotypes of T. reesei mutants. We also successfully reconstructed the three dimensional structure of T. reesei hypha by using focused ion beam SEM technique.

View full abstract

A clinical X-ray CT scanner is an instrument that is used to observe our bodies non-invasively, using the distribution of the linear absorption coefficient. However, X-ray irradiation energy and its spatial resolution are not suitable for the visualization of small objects. Further development and optimization of measurement techniques have led to the development of an X-ray micro-CT scanner for the visualization of small specimens with diameters of a few mm. A technique called refraction-contrast imaging was developed to facilitate the visualization of soft tissue structures, and has become more widely used over time. In this review, the history of X-ray imaging is briefly recounted, and the applicability of these techniques to plant biological research is summarized. In addition, we introduce the authors' works related to these techniques.

View full abstract

Analyses of cellular and subcellular dynamics in living cells and tissues with their three-dimensional (x, y, z) structural information are superior to those with single-plane information. Two-photon spinning disk confocal microscopy is a good method for taking spatially and temporally high-resolution images for reconstruction of three-dimensional view. By using this method, we expect elucidation of dynamics of intracellular structures in a tissue, such as microtubule dynamics in cells inside of a root apical meristem.

View full abstract

To elucidate the regulatory mechanism of division plane establishment is necessary for understanding the development of multicellular organism. In plant cells, the division site, the cortical region where cell plate fuses with the parental cell walls, is pre-determined before nuclear division and the preprophase band marks the site in prophase. Here, I review the research on the development and function of preprophase band based on historical background of our research.

View full abstract

Ever since Darwin’s pioneering research, a major challenge in the field of evolutionary biology has been to understand the regulatory mechanisms that give rise to developmental variation during the course of evolution. Leaf development has been characterized in several species, making the leaf a model organ for analyzing the mechanisms underlying natural morphological variation in plants. In this review, I summarize key findings mainly in the gene regulatory network in leaf development of Arabidopsis thaliana and our recent study investigating the natural variation of leaf shape seen in tomato and relative species. I discuss these issues to understand the molecular mechanism underpinning leaf morphological evolution and to obtain insights into the future direction of plant evolutionary developmental biology.

View full abstract