Doctor of Philosophy

dissertationElectron microscopy can visualize synapses at nanometer resolution, and can thereby capture the fine structure of these contacts. However, this imaging method lacks three key elements: temporal information, protein visualization, and large volume reconstruction. For my dissertation, I developed three methods in electron microscopy that overcame these limitations. First, I developed a method to freeze neurons at any desired time point after a stimulus to study synaptic vesicle cycle. Second, I developed a method to couple super-resolution fluorescence microscopy and electron microscopy to pinpoint the location of proteins in electron micrographs at nanometer resolution. Third, I collaborated with computer scientists to develop methods for semi-automated reconstruction of nervous system. I applied these techniques to answer two fundamental questions in synaptic biology. Which vesicles fuse in response to a stimulus? How are synaptic vesicles recovered at synapses after fusion? Only vesicles that are in direct contact with plasma membrane fuse upon stimulation. The active zone in C. elegans is broad, but primed vesicles are concentrated around the dense projection. Following exocytosis of synaptic vesicles, synaptic vesicle membrane was recovered rapidly at two distinct locations at a synapse: the dense projection and adherens junctions. These studies suggest that there may be a novel form of ultrafast endocytosis

Nano-fEM: Protein localization using photo-activated localization microscopy and electron microscopy

pre-printMapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immunofluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins

Protein localization in electron micrographs using fluorescence nanoscopy

Journal ArticleA complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immuno-electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies, and the inaccessibility of the antigens. Here, we describe a method for correlative fluorescence electron microscopy (fEM) for the localization of proteins in electron micrographs. Proteins tagged with Citrine or tdEos were expressed in Caenorhabditis elegans, fixed and embedded. Tagged proteins were imaged from ultrathin sections using stimulated emission depletion microscopy (STED) or photoactivated localization microscopy (PALM). Fluorescence was correlated with organelles imaged in electron micrographs from the same sections. These methods were used to successfully localize histones, a mitochondrial protein, and a presynaptic dense projection protein in electron micrographs

Decay of the Mediator Particle at Threshold

A light mediator particle is often predicted in the dark sector scenario, which weakly interacts with the standard model (SM) particles. The weakness of the interaction is usually described by a small coupling; however, the small coupling does not always guarantee the weakness of the interaction. When the mass of the mediator particle lies in a threshold region, the so-called threshold singularity may emerge, and then the perturbative calculation fails. This singularity causes several effects, e.g., the mixing between the mediator particle and bound states, the Sommerfeld effect on the final state of the mediator particle decay, etc. Taking the minimal model of the vector mediator particle decaying mainly into the SM particles as an example, we develop a method to describe the singularity quantitatively. We also calculate some physical quantities using this method, such as the lifetime of the mediator particle, and find that those could be significantly altered compared with the result of the perturbative calculation.Comment: 25 pages, 6 figure

Static magnetic moments revealed by muon spin relaxation and thermodynamic measurements in quantum spin ice Yb2_2Ti2_2O7_7

We present muon spin relaxation ($\mu$SR) and specific-heat versus temperature $C(T)$ measurements on polycrystalline and single-crystal samples of the pyrochlore magnet Yb$_2$Ti$_2$O$_7$. $C(T)$ exhibits a sharp peak at a $T_\mathrm{C}$ of 0.21 and 0.26~K for the single-crystal and polycrystalline samples respectively. For both samples, the magnetic entropy released between 50~mK and 30~K amounts to $R\ln2$ per Yb. At temperatures below $T_\mathrm{C}$ we observe a steep drop in the asymmetry of the zero-field $\mu$SR time spectra at short time scales, as well as a decoupling of the muon spins from the internal field in longitudinal magnetic fields of $\leq0.25$~T for both the polycrystalline and single-crystal samples. These muon data are indicative of static magnetic moments. Our results are consistent with the onset of long-range magnetic order in both forms of Yb$_2$Ti$_2$O$_7$.Comment: 6 pages, 4 figures, accepted to PR

Studies on the Body Surface Area of Japanese

Surface area of 201 Japanese (both sexes) from infancy to old age selected statistically by sex, age and body shape were measured and the regional rates calculated and discussed. The results are as follows. 1. There was no difference between either side of the body for age or sex. 2. The rate of head, face and neck decreased while that of the lower extremities increased with aging. 3. After adolescence regional rates could be differenciated between the sexes. The rates of the thigh of the female were considerably higher than that of the male. 4. As for body shape, rate of the head, face and neck tended to lower toward obesity and rose toward slenderness. The rates of the trunk, upper and lower extremities revealed the opposite tendency to the former. 5. These results vary little as compared to other studies done on Japanese. However, compared to a German or an American revealed a higher rate for limbs. The rate was definetely lower for the Westerner

Seasonal Variation of Energy Metabolism

Seasonal variation of the energy metabolism of Japanese was measured on the following items. 1) Seasonal variation of the basal metabolism by sex and age-group, and by intensity of work on some age-groups. 2) Seasonal variation of the rest metabolism by age-group on male. 3) Seasonal variation of work metabolism and relative metabolic rate (R. M. R.). 4) As supplement, influence of menarche on variation of basal metabolism. In general, it is shown that the energy metabolism of Japanese variates under the influence of seasonal variation of Japanese climate, as basal metabolism of adult is low in summer and high in winter. But basal metabolism of infant shows two peaks in a year. And rest metabolism of infant and school age children have different seasonal variation from those of adult and aged. On work metabolism, aged subjects showed more variance than other age-groups

High-speed simulation of PCB emission and immunity with frequency-domain IC/LSI source models

Some recent results from research conducted in the EMC group at Okayama University are reviewed. A scheme for power-bus modeling with an analytical method is introduced. A linear macro-model for ICs/LSIs, called the LECCS model, has been developed for EMI and EMS simulation. This model has a very simple structure and is sufficiently accurate. Combining the LECCS model with analytical simulation techniques for power-bus resonance simulation provides a method for high-speed EMI simulation and decoupling evaluation related to PCB and LSI design. A useful explanation of the common-mode excitation mechanism, which utilizes the imbalance factor of a transmission line, is also presented. Some of the results were investigated by implementing prototypes of a high-speed EMI simulator, HISES. </p