The imaging technique of the location of molecules in cells and tissues had remarkably advanced life science. Nowadays, improved techniques are required for image information that is quantitative, high resolution, 3D or in a wide area. As such studies this symposium introduced the electron microscope imaging and the automatic image analysis of a wide area, the analysis of elements in the plant tissues with an isotope microscope or SIMS (Secondary Ionization Mass Spectrometer), as well as high-resolution imaging of alive tissues using a 2 photon microscope.
Transmission electron microscopy (TEM) is important for ultrastructure observation for organelles, cells and tissues. However, it is unsuitable for comprehensive analyses. We have constructed a high resolution TEM image acquisition system to take a giga-pixel electron micrograph. An auto-acquisition TEM system can control TEM system such as a X-Y stage, electron beam and a CCD camera by remote PC, and acquired continuous electron micrographs. We succeed in records more than 30 thousand images. We developed image-tiling program for TEM pictures, and several thousands of pictures were automatically merged. In this article, we describe the mechanism of high resolution TEM image acquisition system and the applications by using high-pressure freezing method.
Recent advances in imaging equipment have enabled the acquisition of many kinds of bioimages in huge numbers. With the acquisition of such imagery, computer assistance becomes increasingly important for image inspection. To provide an automated and versatile bioimage classification system, we have developed an active learning algorithm combined with a genetic algorithm and self-organizing map named Clustering-Aided Rapid Training Agent (CARTA). Using CARTA, similar images can be drawn from many images. Applying this feature of CARTA, we are developing a framework for the detection of similar cellular architectures in wide-field fluorescence microscopic images. In this article, we describe an example case of semi-automatic detection of stomatal regions from a fluorescence microscopic image of Arabidopsis leaf surface cell contours.
Plants use inorganic minerals for nutrition from soil. To understand inorganic minerals transport pathway is very important for not only plant science but also food crisis. To identify locate and quantify elemental distribution in plant cells, which required high resolution and high sensitive method. Secondary ion mass spectrometry (SIMS) is an analytical technique, which is a variety of fields spanning from the material science over biology to geochemistry, cosmochemistry and plant science. A new-generation SIMS instrument, Isotope Microscope System (IMS), has unique characteristics for the 3D visualization of microscopic regions of materials through high-precision imaging of elemental isotopes. IMS allows visualization of stable isotopes at ultra-high sensitivity and highresolution, and now is useful also for biological applications. SIMS is required high vacuum therefore live cells cannot be analyzed. Thus it is important to sample preparation which cells must be preserved as close as possible to their native state. This review focuses on element detection equipment about X-ray method, ICP-MS, SIMS and explained about most suitable sample preparation of plant material for SIMS analysis. SIMS equipment allowed subcellular element distribution in plant cells. I conclude that IMS and NanoSIMS are powerful tools for visualization of plant nutrient dynamics in subcellular level.
A high lateral resolution secondary ion mass spectrometer (NanoSIMS) is a powerful tool for the analysis of biological samples. It provides direct-imaging of elements and isotopes with subcellular scale and low detection limit. In combination with stable isotope tracers, quantitative imaging has been used to investigate various biological activities, such as carbon and nitrogen transfer and metabolism in animal and bacterial cells. This mini review describes some basic information of SIMS and applications of elemental imaging in biological studies using NanoSIMS.
In vivo live-cell imaging is an essential for understanding biological phenomena. In biological science, recent advances in imaging techniques using various fluorescence probes revealed dynamic cellular/extracellular events as they occur in real time at subcellular level. Two-photon excitation microscopy provides non-invasive tool for deep imaging in living organisms. We have been trying to deep imaging in plant tissues, using two-photon excitation microscopy to study the biological phenomena in vivo. In this review article, we summarize the characters of two-photon microscopy and advantages for deep imaging of plant tissues in vivo.
For the field of neuroscience, laser-scanning florescence microscopy utilizing two-(or multi-) photon excitation process (two-(multi-)photon excitation laser scanning microscopy, two-(multi-)photon microscopy) has become widely used as an essential tool for biological and medical research including cancer, and immune researches. Especially, “in vivo” two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, that enabled us to identify individual fluorescent beads of which diameter is 170 nm; smaller than classical PSF width. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.
In homosporous ferns, gametophytes are cordate-thalloid (terrestrial) in many taxa but can also be strap- or ribbon-like (epiphytic), filamentous (epiphytic), and tuberous (subterranean) in some taxa. Recently developed long-term observational techniques of the same individual gametophyte during growth have led to a new classification of development types for planar gametophytes. We recognize five basic types (Lygodium-, Elaphoglossum-, Anemia-, Colysis-, and Vittaria-types). The five types reflect different combinations of the three meristems: apical cell-based, marginal and multicellular meristems. Cordate, spatulate, and asymmetrically cordate gametophyes are formed by the apical cell-based and subsequently formed multicellular meristems (Lygodium-, Elaphoglossum-, and Anemia-types). Irregularly branched, ribbon-like gametophytes are characterized by occurrence of the marginal meristem. During the marginal meristem phase, the gametophyte branches to form many lobes by cessation of the marginal meristem. In the Colysis-type, the marginal meristem phase occurs between the apical cell-based and the multicellular meristem phases, and in the Vittaria-type, the multicellular meristem phase is absent. The ability of many lobe formation leads to clone formation, resulting in slow growing and long-life of the gametophyte.
Plant development has been evaluated at various developmental stages, from the early steps of embryogenesis to flowering. In most reports, transcription factors have been thought to play a master regulatory role in the complex networks orchestrating organogenesis. Although these efforts have increased our understanding of several major developmental pathways, our understanding of the relationships between metabolism and development remains limited. Recently, we identified a straightforward relationship linking carbohydrate metabolism and organogenesis. We found that plant development, particularly the reactivation of cell cycling after germination and the transition from heterotrophic to autotrophic growth, are highly dependent on sucrose availability. In the case of Arabidopsis thaliana, an oilseed species, we characterized the importance of cytosolic inorganic pyrophosphate hydrolysis for the success of the above transition and appropriate execution of postembryonic developmental programs. While this unprecedented and unique discovery has addressed fundamental issues concerning the biological role of the proton-pyrophosphatase (H+-PPase), it has also raised questions regarding the link between metabolism and development. Here, we summarize our present knowledge of key steps in the mobilization of storage lipids and their impact together with H+-PPase during the heterotrophic-autotrophic growth transition.
Plant cells divide by formation of the cell plate. The cell plate is formed by the cytokinetic apparatus phragmoplast. The phragmoplast is a cytoskeleton-membrane complex, which contains many microtubules. Although expansion of the phragmoplast drives growth of the cell plate towards the plasma membrane, the mechanism is unclear. The author’s group demonstrated microtubule organization and dynamics in the phragmoplast, and proposed the mechanism how the phragmoplast expands. I will describe the mechanism of phragmoplast expansion in this review.
Cells in clonally different lineages coordinate proliferation within a developing organ to ensure species-specific organ size and shape, and signaling between cells generally plays a pivotal role in this process. In the field of developmental biology, examining the molecular details and dynamics of the signaling remains a challenge. Previous studies have suggested that the size and shape of a leaf are determined through coordinated proliferation between epidermal and mesophyll cells, possibly through inter-celllayer signaling. However, the signaling mechanism remains largely unexplored because, until recently, little was known regarding the signaling molecules involved in this process. ANGUSTIFOLIA3 (AN3) in Arabidopsis thaliana encodes a transcriptional co-activator with homology to human synovial sarcoma translocation protein. Kawade et al. (2013) reported that AN3 is a signaling molecule that mediates inter-cell-layer signaling for the coordinated proliferation of leaf cells. AN3 is produced specifically in mesophyll cells and moves into epidermal cells, where it participates in transcriptional control for cell proliferation in destination cells. The identification of the signaling molecule involved in coordinated proliferation of leaf cells allows us to directly investigate the functional impact of inter-cell-layer signaling on leaf development. Interfering with AN3 movement across cell layers disrupts the coordinated proliferation of leaf cells. Leaf size and shape are also impaired in this situation, demonstrating that AN3 signaling itself is indispensable for normal leaf development. Based on these findings, Kawade et al. (2013) presented a novel model emphasizing the role of mesophyll cells as a signaling source for coordinated proliferation of leaf cells, as well as the regulation of leaf size and shape.
The Podostemaceae are aquatic angiosperms of the tropics and subtropics that have remarkably specialized morphology and are adapted to rheophytic habitats that extreme torrential floods. The subfamily Podostemoideae is devoid of typical shoot apical meristem and form an aggregate of leaves arising from the root. By contrast, the basal subfamily Tristichoideae has a shoot-like structure called “ramulus”. The ramulus branches several times. The new ramulus is initiated as extra-axillary buds of immediately older ramulus. After forming ramulus branches, the main ramulus become determinate in growth, associated with the loss of the apical meristem. This manner of branching system is the sympodial branching which is often found in other angiosperm shoots. The sympodial branching pattern in Tristichoideae resembles that in the subfamily Weddellinoideae. The “ramulus” of the Tristichoideae and the “leaf” Podostemoideae share an organogenetic pattern in which new organs initiate at the base of organs, irrespective of differences in the kinds of organs. Additionally, Podostemaceae has dimorphic chloroplasts in the epidermis which have not been reported in any other aquatic angiosperms. This suggests that the dimorphism represents adaptation to the unique habits of Podostemaceae.