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関連する概念動画

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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Overview of Electron Microscopy01:25

Overview of Electron Microscopy

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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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Scanning Electron Microscopy01:07

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A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.
Fundamental Principles
Accelerated...
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Transmission Electron Microscopy01:15

Transmission Electron Microscopy

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In 1931, physicist Ernst Ruska—building on the idea that magnetic fields can direct an electron beam just as lenses can direct a beam of light in an optical microscope—developed the first prototype of the electron microscope. This development led to the development of the field of electron microscopy. In the transmission electron microscope (TEM), electrons are produced by a hot tungsten element and accelerated by a potential difference in an electron gun, which gives them up to 400...
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Immunogold Electron Microscopy01:20

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Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
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Super-resolution Fluorescence Microscopy01:37

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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高解像度冷凍電子顕微鏡用のバイオアクティブ機能化された単層グラフェン

Nan Liu, Jincan Zhang, Yanan Chen

  • 1Collaborative Innovation Center of Quantum Matter , Beijing 100871 , China.

Journal of the American Chemical Society
|February 7, 2019
PubMed
まとめ

新しい機能化されたグラフェン膜 (FGM) のグリッドは,冷凍電子顕微鏡 (cryo-EM) の試料の準備を改善します. これらのグリッドはヒスティジンで標識されたタンパク質に特異的に結合し,デナチュレーションを減らし,高解像度の構造的決定を可能にします.

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科学分野:

  • 構造生物学
  • バイオ物理学
  • 材料科学

背景:

  • 単粒子の冷凍電子顕微鏡 (cryo-EM) は,分子レベルの生物学的洞察に不可欠です.
  • 試料の準備,特にガラス化氷の埋め込みは,冷凍EMのボトルネックです.
  • 従来の方法は,空気と水のインターフェイスでタンパク質の 変性化と方向偏移につながる.

研究 の 目的:

  • 試料の準備を改善するために新しい冷凍-EMグリッドを開発する.
  • 伝統的な冷凍-EMサンプルマウント技術の限界を克服するために.
  • クリオ・エム構造研究の再現性と解像度を向上させる.

主な方法:

  • 生物活性リガンド機能化された単層結晶グラフェン膜 (FGM) の設計と製造.
  • ヒスティジン (His) -タグされたタンパク質に対する特定の結合親和性を有するFGMグリッドを使用します.
  • タンパク質複合体のイメージングと構造再構築のためのFGMグリッドの応用

主要な成果:

  • FGMグリッドはHISタグのタンパク質と複合体に 特定の結合を示しています
  • グリッドは低画像背景を提供し,選択的に20Sプロテアソームを固定します.
  • 20Sプロテアソームの3D再構築は,FGMグリッドを使用してほぼ原子解像度を達成しました.

結論:

  • 機能化されたグラフェン膜グリッドは,冷凍-EM試料の準備のための堅固なソリューションを提供します.
  • FGMグリッドは再現性を高め,変性化を軽減し,構造的決定の効率を向上させます.
  • このアプローチは,高解像度の冷凍-EM構造生物学を大幅に進める可能性があります.