Brown algae including Saccharina and Undaria show the largest photosynthetic organisms in coastal area. All species possess multicellularity, and there are several varieties of body plan; i.e. uniseriate and multiseriate filamentous thalli, and the complex multicellularity. Brown algal cells have tiny cytoplasmic connections, plasmodesmata, with 10-20 nm diameter. Also in land plants, plasmodesmata are observed within the septum cell wall and they are formed during the cell plate formation. When plasmodesmata are created on the cytokinesis, they are called primary plasmodesmata. Cytokinesis and existence of primary plasmodesmata in brown algae had been unclear. We examined the process of cytokinesis using electron tomography and revealed the appearance of plasmodesmata during cytokinesis. Cytokinesis of brown algae proceeds as follows; cytokinetic plane is determined by crossing region of microtubules from centrosomes, Golgi derived vesicles and flat cisternae fuse each other to form new cell partition membrane, and then centrifugally growing new cell partition membrane reaches to the mother cell wall.
Electron tomography is a powerful technique to visualize and quantitatively analyze ultrastructure within cells. Three-dimensional volume is reconstructed from s series of images that is obtained by sequentially tilted sample. The combination of high-pressure freezing and electron tomography was applied to analysis of actins and microtubules in preprophase band of onion cotyledon epidermal cell. The preprophase band is cytokinetic apparatus, which is detected in cell cortex as bundled microtubules. Although it is suggested that actins contribute to organization of microtubules, how actins contribute to organization of the microtubules remains unsolved. To address the question as to how F-actins interact with other preprophase band components requires 3D structural studies at electron microscope level. Electron tomographic analysis depicts that detailed distribution of the microtubules and fine microfilaments, and relationship between them.
Clearing techniques are essential for deep imaging by a microscope. “Scale” which is a clearing technique for animal brain triggers the race to develop next-generation clearing techniques for animal tissues. In the case of clearing techniques for plant tissues, several groups have published similar clearimg methods, however these methods took several days to weeks. We developed a rapidly clearing technique “TOMEI (Transparent plant Organ MEthod for Imaging)” which enables to clear plant samples within 6 hours completely and detect fluorescent protein signals at a depth of approximately 200 micrometers using confocal laser scanning microscopy. We showed that TOMEI could be applied for leaves, flower buds, roots, and root knots in A. thaliana and leaves in O. sativa.
During double fertilization, a unique reproductive strategy in angiosperms, two sperm cells fuse with two distinct female partners, an egg cell and a central cell. The fusion products develop into an embryo and its nutritious tissue, an endosperm, respectively. The difficulty in observing double fertilization process hampers progress of double fertilization research. Recent breakthrough in microscopy techniques to observe living cells and cell manipulation techniques, such as inducible gene expression and laser disruption of specific cells, began to reveal the mechanism of double fertilization in Arabidopsis thaliana. Here, we review current discoveries about the dynamics of double fertilization process and also discuss future views on plant reproduction research based on live imaging techniques.
Mono-innervation by single climbing fibers and segregated dendritic innervation by climbing fibers and parallel fibers are the two distinguishing features of synaptic circuits in cerebellar Purkinje cells. In the process, GluD2 or GluRd2, which is selective to parallel fiber-Purkinje cell synapses, plays key roles in development, maintenance, and regeneration of Purkinje cell circuits through its involvement in the strengthening of synaptic connectivity. This selective strengthening leads to activation of metabotropic glutamate receptor mGluR1 at parallel fiber synapses, which then propels eliminations of surplus climbing fiber synapses from the soma and of ectopic parallel fiber synapses from the proximal dendrite. Through this interplay by GluD2 and mGluR1, two distinguishing features of Purkinje cell circuits develop and maturate.
Podocytes, an essential cellular compartment of the glomerular filtration barrier, present a unique three-dimensional architecture specialized for their function. However, several aspects on three-dimensional morphological aspects of podocytes remain partially understood because they are difficult to reveal using conventional scanning electron microscopy. Here, we adopted FIB-SEM tomography, a powerful tool for analyzing the three-dimensional cellular ultrastructure, to precisely reveal the three-dimensional architecture of podocytes in health, development, and disease.
A survey on electron lens designing methods is presented in two consecutive articles. The present article comprises the first part and discusses the optics aspect of the lens design. In electron optics the electromagnetic potential plays the role of refractive index in ordinary light optics. The electron optics design is to construct an appropriate potential distribution under a strong constraint that it must satisfy the Laplace equation. The basic strategy to realize good lenses has been to have as short a focal length as possible in order to suppress inevitable aberration effects. We explain the designing principles for achieving short focal lengths. An efficient lens design method that combines the optical parameters obtained from the paraxial trajectory calculation and the emittance diagram representation is presented as well. The sequel article will give example designs of short focal length lenses together with the engineering techniques for their implementations.
Neurons and glial cells that are main component of brain tissue are distributed in tree-dimensional space. The space exists even if within tens of micrometers-thickness slices. Sampling bias is a big problem well known when we estimate the number and distribution of cells by information from two-dimensional sections. It might be because that the large size and irregular shaped cells have a greater opportunities counted repeatedly than small and uniform one, respectively. Thus, to obtain rigorous quantitative data from two-dimensional specimens, it is necessary to estimate number and density of cells in particular brain regions by the unbiased stereological method, the disector, and a tree-dimensional counting rule. Here, we would like to show useful quantitative analyses in light and electron microscopic specimens by the disector.
We have demonstrated quantitative annular dark-field (ADF) imaging for crystal structure analysis. Quantitative ADF contrast is given as a scattering intensity normalized by an incident probe current. We have developed a quantification procedure, in which the non-linear response of ADF detection system is corrected. Observed ADF quantitative contrast is in agreement with that of simulated images, and the quantitative ADF imaging allows us to directly count the number of graphene layers. We have also investigated how accurately atomic-resolution ADF images match between experiments and simulations. An appropriate effective source distribution function should be implemented in simulations, and we found the linear combination of Gaussian and Lorentzian functions well reproduces experimental profiles.