KENBIKYO
Online ISSN : 2434-2386
Print ISSN : 1349-0958
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Displaying 1-8 of 8 articles from this issue
Feature Articles: Biological CLEM Today: Connecting Light and Electron Microscopy for Advanced Applications
  • Keisuke Ohta
    2024 Volume 59 Issue 3 Pages 98
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
    JOURNAL RESTRICTED ACCESS
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  • Satoshi Kikkawa, Ryo Nitta, Keisuke Ohta
    2024 Volume 59 Issue 3 Pages 99-104
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
    JOURNAL RESTRICTED ACCESS

    Correlative Light and Electron Microscopy (CLEM) is a set of methods for observing the same region of a sample using both light and electron microscopy. These methods are used for various purposes, such as simultaneous observation with two modalities, correlating cell type identification, or pinpointing regions identified by light microscopy for electron microscopy observation. The targets of CLEM also range from protein structures to tissues, requiring a variety of techniques tailored to specific needs. This article provides an overview of the CLEM workflow and offers a compass for selecting appropriate methods to achieve desired outcomes. While there are numerous reviews on CLEM using resin-embedded specimen by sections and 3D electron microscopy, this review focuses specifically on cryo-CLEM, which aims to visualize protein structures within specific intracellular regions.

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  • Shuichi Hayashi, Nobuhiko Ohno
    2024 Volume 59 Issue 3 Pages 105-109
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
    JOURNAL RESTRICTED ACCESS

    Correlative light and electron microscopy (CLEM) is an imaging technique for analysing the same specimen with light microscopy (LM) and electron microscopy (EM). This technique combines the advantages of the two imaging modalities to correlate wide-field and/or functional information obtained by LM with ultrastructure obtained by EM. Recent advances in serial imaging techniques using scanning EM (SEM) allow us to perform three-dimensional (3D)-CLEM. In biological applications of CLEM, various approaches have been developed depending on what we observe and how we label the target. In this article, we show a CLEM workflow using serial block-face SEM on brain tissues, in which a particular subtype of neurons is labelled with fluorescent protein. Endogenous structures such as blood vessels and cells are used as landmarks on brain tissues without immunostaining, which maintains optimal tissue conditions for EM observation. We illustrate how this workflow using confocal laser scanning microscopy and SBF-SEM is applied to the study of hippocampal synapses.

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  • Naomi Kamasawa
    2024 Volume 59 Issue 3 Pages 110-113
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
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    Correlative Light and Electron Microscopy (CLEM) is a technique well-suited for investigating the relationship between cellular function and fine structure. It involves recording cellular activities using advanced fluorescence microscopy techniques and analyzing the three-dimensional ultrastructure of the same cells using Volume Electron Microscopy (vEM). Additionally, vEM images allow for the extraction and examination of information about internal organelles and/or surrounding structures of the targeted cells, enabling further exploration of cellular functions. While vEM data contains vast amounts of biological information, effectively extracting useful information from the complex, condensed black-and-white images requires the use of automation using AI tecnologies. This paper presents examples of functional measurements of neuronal cells using two-photon microscopy and the investigation of synapse structure from correlated vEM data.

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  • Isei Tanida, Junji Yamaguchi, Chigure Suzuki, Soichiro Kakuta, Yasuo U ...
    2024 Volume 59 Issue 3 Pages 114-118
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
    JOURNAL RESTRICTED ACCESS

    Advances in fluorescence microscopy, including super-resolution microscopy, and semi-in-lens type scanning electron microscopy have expanded the field of imaging in biological materials. As a result, there is an increasing demand for more precise analytical methods in correlative light and electron microscopy (CLEM). In-resin CLEM, which involves simultaneous observation and correlative analysis of the same ultrathin section of resin-embedded biological materials using both fluorescence and electron microscopes, has improved correlation accuracy. Ideally, in-resin CLEM should use epoxy resin due to its superior ultrastructural preservation. However, many fluorescent proteins and dyes lose their fluorescence due to the autofluorescence of epoxy resin itself and chemical treatments such as osmium tetroxide staining. Various approaches have been taken to solve these problems, and recently, ‘Immuno in-resin CLEM” at the tissue level was performed by applying immunological reactions.

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Review
  • Toshiaki Tanigaki, Tetsuya Akashi, Takaho Yoshida, Ken Harada, Kazuo I ...
    2024 Volume 59 Issue 3 Pages 119-126
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
    JOURNAL RESTRICTED ACCESS

    The analysis of the magnetic structure of materials and the associated spin configurations is important not only in the fields of solid state physics, inorganic chemistry, and spintronics, but also in other areas such as materials science and engineering. Atomic-level analysis of local magnetic fields has been reported using electron energy-loss spectroscopy with aberration-corrected electron microscopy and differential phase contrast measurements. However, it has been difficult to directly observe the magnetic field at the atomic level when analyzing the magnetic field of a sample with multiple elements responsible for the magnetic field or with a thickness distribution. In this paper, we report the development of a pulsed magnetic field application system, post-digital aberration correction, and a multislice simulation including magnetic field based on an aberration-corrected high-voltage holography electron microscope. And the results of the successful observations of the phase distributions reflecting the magnetic field on the (111) lattice planes formed by the opposite spin ordering in ferrimagnetic double-perovskite oxides (Ba2FeMoO6) are described.

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Lectures
  • Masataka Yanagawa
    2024 Volume 59 Issue 3 Pages 127-132
    Published: December 30, 2024
    Released on J-STAGE: January 17, 2025
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    Understanding the mechanism of drug-activated membrane receptor signaling in living cells is crucial for advancing molecular pharmacology. While electron microscopy has provided detailed structural insights into these receptors and associated signaling molecules, the precise cellular locations and dynamics of these complex formation and dissociation upon drug stimulation are still not fully understood. Single-molecule imaging techniques, including total internal reflection fluorescence (TIRF) and thin-layer oblique illumination (HILO) microscopy, allow for quantification of diffusion dynamics and molecular interactions in living cells with high positional accuracy (8–30 nm) and temporal resolution (30~50 ms). This approach has revealed that the clustering of membrane receptors within lipid domains significantly influences signal transduction. This article reviews the fundamental optical microscopy techniques, cell sample preparation methods, and single-molecule tracking analysis. It also highlights the latest technological advances in single-molecule imaging and discusses potential applications in pharmacology and drug discovery.

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