フランス高等教育・研究省に感謝しております規則 Industrielles ド形成パー ラ抜き (CIFRE) と金融支援 IFP エネルギーヌーヴェル。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J Struct Biol. 116 (1), 71-76 (1996).</li><li>Align RGB planes. ImageJ Available from: <a target="_blank" href="https://ImageJ.net/Align_RGB_planes">https://ImageJ.net/Align_RGB_planes</a> (2018)</li><li>MessaoudiI, C., Boudier, T., Sorzano, C., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinf. 8 (1), 288 (2007).</li><li>Saxton, W. O., Baumeister, W., Hahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+imperfect+two-dimensional+crystals.">Three-dimensional reconstruction of imperfect two-dimensional crystals.</a> Ultramicroscopy. 13 (1-2), 57-70 (1984).</li></ol>

著者が明らかに何もありません。

本稿の目的は、透過トモグラフィによる化学の 3 D のマップを取得する方法について説明することです。このプロトコルは完全にオリジナルと著者らによって開発されました。
EFTEM トモグラフィ前述のようにいくつかの欠点があります: フィルターされた画像を取得するために必要な長い露出時間による電子ビーム耐性は、(i) のみサンプルが分析できます。(ii) EFTEM トモ?...

機能性材料の特性は 3 D パラメーターに依存しています。完全に彼らの特性を理解し、その機能を強化、形態および 3 D で化学の分布を分析することが重要です。電子断層撮影法1 (ET) は、ナノメートル スケールの2,3でこの情報を提供する最高のテクニックのひとつです。それは広い角度範囲にサンプルを回転と角度ステップごとに 1 つの画像を記録から成っています。得られた傾きのシリーズを使用して、ラドン変換4,5に基づく数学的なアルゴリズムを使用して、サンプルのボリュームを再構築します。3 D のサンプルをモデルしのような粒子局在6とサイズ分布73D パラメーターを定量化、毛穴の位置とサイズ分布8等は、ボリュームの灰色レベルを選択すること
一般的には、ET は、できればどちらの方向に 70 ° 以上の最大の可能な角度にサンプルを傾けることによって電子顕微鏡で実行されます。各傾斜角に成型、画像の傾きシリーズ サンプルの投影が記録されます。その傾きの系列を整列し、セグメント化と定量化がサンプルのボリュームを再構築するために使用します。+90 °、-90 ° からサンプルを回転ことはできません、ために、盲検角度による直交軸9に沿って異方性解像度があります再構成ボリューム。
ET は、イメージングの異なるモードで実行できます。触媒サポート複雑な形状または明視野 TEM モード (BF TEM) は非晶質材料、生体試料、高分子を研究に使用されます。画像解析は、コンポーネント10 (高密度になります詳細ライターよりも暗い、すなわち密度の低いコンポーネント) の密度を特徴付ける灰色レベルの分化に基づいています。高角環状暗視野 TEM モード (関数幹) をスキャンする際に、結晶性試料を分析する使用されます。信号は、原子番号の機能として化学情報を提供しますサンプルの重いコンポーネントが明るく表示される、ライター 1 つ9。収集材料11、およびエネルギーによって放出される x 線エネルギー分散型 x 線分光 (EDX) のような他のモード イメージング モード (EFTEM)12,13をフィルタ リング、3 D の化学的分布を評価することができるもサンプル。
EFTEM イメージング、電子顕微鏡を用いた電子エネルギー分析器 2D 化学マップを記録できます。分光計は、彼らのエネルギーの機能として電子を分散させることによって磁気プリズムとして機能します。イメージは、特定の原子との相互作用から失われたエネルギーによって電子によって作成されます。同じ 2 D 化学マップが異なる傾きの角度、化学予測のシリーズを取得すると、傾斜で計算される場合 3 D 化学ボリュームを再構築するを使用できます。
すべての材料は、透過トモグラフィによる分析できます。テクニックは、弱いまたは不規則材料サンプルの予約されています。それにもかかわらず、他のイメージング技術を使用してときに区別し非常ににくい軽元素の分析に使用できます。さらに、信頼性の高い 2 D 化学マップを得るためには、材料の厚さは、材料14を介して電子の平均自由行程以下に必要です。この条件下で最大の単一電子単一原子との相互作用を持っていることの確率です。2 つのメソッドは、2 D 化学マップの計算に使用されます。1 番目は、最もよく使われるイオン化エッジ13後分析、および 3 分の 1 の下の要素のイオン化エッジの前に 2 つのフィルターが適用されたエネルギー ウィンドウを記録する場所「3 windows 法」をです。最初の 2 つの画像を用いて推定して、3 番目のウィンドウの位置から力法を使用して、それから差し引かれる背景を推定します。得られた画像は、3 D サンプル ボリュームの分析元素分布の投影です。2 番目のメソッドは、「ジャンプ比;」と呼ばれる前に、と後の 2 つだけエネルギー フィルター画像を使用してイオン化エッジ。この方法は質的、最終的なイメージはこれらの 2 つの画像間の比率を実行するだけで計算され、背景エネルギーの変化については考慮しません。
EFTEM ET と組み合わせることで、フィルター選択されたエネルギーの解析的トモグラフィーを得ることができます。EFTEM トモグラフィーとアトム プローブ断層レントゲン写真撮影 (APT) は、相補的な技術です。APT、に比べて EFTEM 断層レントゲン写真撮影は複雑な前処理を必要としない非破壊特性解析です。ユニークなナノ粒子の様々 な特徴を実行する使用できます。EFTEM トモグラフィーは、APT は、それらを測定する非常に少なくともレーザー支援で必要としながら絶縁材料を分析できます。EFTEM 断層が低解像度で適切に動作しながら、原子スケールの APT が実行されます。透過トモグラフィ実験中にビームの劣化に抵抗するサンプルに対してのみ適切であります。すべての傾斜角度でのすべてのフィルターが適用された画像を記録するには、サンプルは 2 時間限りの電子ビームを公開できます。さらに、2 D マップで最大の化学信号を記録する高いビーム強度で長い博覧会の期間は、必要かもしれません。このような条件でビームの敏感なサンプル抜本的な形態学的・化学的変化に苦しみます。したがって、電子ビームにサンプル感度の正確な測定は、実験前に確立されなければなりません。さらに、透過トモグラフィは空間的な位置およびサンプル.に存在する元素の性質を決定する必要に応じてできるだけ多くの断層を記録の結果それにもかかわらず、EFTEM トモグラフィーは、触媒アプリケーションをモデル化するための新たな洞察を与えるための触媒担体などのサンプルの 3 D 化学配分に関する重要な情報を提供できます。
今日それはエネルギー間隔を選択することができます専用のソフトウェアを使用することが可能、記録エネルギー ウィンドウ画像をフィルター処理し化学物質が異なる角度でマップを計算します。サンプルでは、追跡、焦点を当て、および透過モードでフィルター処理された画像の記録を傾斜できます。2D 化学マップを計算できると、フルティルト シリーズ配置できますが、反復的なアルゴリズムを使用して計算化学のボリュームとシリーズがセグメント化と定量化された15,16をすることができます最後に。

<table><tbody><tr><td>JEOL 2100f</td><td>JEOL</td><td></td><td>Electron microscope</td></tr><tr><td>Tridiem Gatan Imaging Filter (GIF)</td><td>Gatan</td><td></td><td>Post colum energy filter</td></tr><tr><td>Digital micrograph</td><td>Gatan</td><td></td><td>Software</td></tr><tr><td>Gatan EFTEM tomography plugin</td><td>Gatan</td><td></td><td>Dedicated software to record filtered tilt series for EFTEM tomograohy</td></tr><tr><td>Tomoj</td><td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td><td></td><td>Free software developed by Currie Institute in Paris, France for electron tomography</td></tr><tr><td>EFTEM-Tomoj</td><td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td><td></td><td>Free software developed by Currie Institute in Paris, France , for EFTEM imaging</td></tr><tr><td>Imod</td><td>http://bio3d.colorado.edu/imod/</td><td></td><td>Free software developed by University of Colorado, USA for electron tomography</td></tr><tr><td>Imagej</td><td>https://imagej.nih.gov/ij/</td><td></td><td>Free software developed by National Institute of Mental Health, Bethesda, Maryland, USA for images treatment</td></tr><tr><td>Merge channels</td><td>https://imagej.net/Color_Image_Processing</td><td></td><td>Fonction in Imagej allowing to give different colors to volumes while they are overlapped</td></tr><tr><td>3D Slicer</td><td>https://www.slicer.org/</td><td></td><td>Free software developed by a large consortium lead by Ron Kikinis , Harvard Medical School, Boston, MA, SUA</td></tr><tr><td>Chimera</td><td>https://www.cgl.ucsf.edu/chimera/</td><td></td><td>Free software developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco,for data segmentation, cuatification and visualisation of 3D models</td></tr><tr><td>silica alumina support of catalyst</td><td>IFPEN</td><td></td><td>sample prepared for eleboration of this protocol</td></tr><tr><td>titania alumina support of catalyst</td><td>IFPEN</td><td></td><td>sample prepared for eleboration of this protocol</td></tr><tr><td>alcohol</td><td></td><td></td><td></td></tr><tr><td>water</td><td></td><td></td><td></td></tr><tr><td>Au nanoparticles of 5 nm</td><td>BBI Solutions</td><td></td><td></td></tr><tr><td>Holey carbn film 200 mesh microscopy grid</td><td>Agar</td><td></td><td></td></tr><tr><td>EDX sepctrometer</td><td>Oxford Instruments</td><td></td><td></td></tr></tbody></table>

obtaining 3d chemical maps by energy filtered transmission electron microscopy tomography

エネルギー フィルター透過電子顕微鏡トモグラフィー (EFTEM 断層レントゲン写真撮影) は、ナノメートル スケールでの材料の三次元 (3 D) 化学マップを提供できます。EFTEM 断層撮影は、他のイメージング技術を使用して区別するために非常に困難な化学元素を区切ることができます。ここで記述されている実験のプロトコルは、化学的分布と材料の形態を理解する化学 3 D のマップを作成する方法を示しています。データ分割の準備手順、サンプルが掲載されています。このプロトコルは、ナノメートルのサンプルの化学要素の 3次元分布解析を許可します。ただし、現在、3 D 化学マップのみ生成されることをサンプル フィルターされた画像の記録は、強烈なビームに長い露出時間を必要とするので、機密性の高い、ビームではないです。プロトコルは、2 つの異なる異種触媒担体の成分の化学分布の定量化に適用されました。最初の研究では、アルミニウム、チタン酸化チタン アルミナ サポートでの化学の分布を調べた。スイング pH メソッドを使用して調製します。第二に、ソル粉と機械的混合法を使用して作製したシリカ-アルミナ サポート中のアルミニウムおよび si の化学の分布を調べた。

参照13に、このプロトコルのアプリケーションの例を示します。EFTEM トモグラフィ チタニア アルミナ触媒担体の分析に用いる。水素化脱硫 (HDS) のようなアプリケーションで、MoS2ナノ粒子の活動的な段階の触媒の活性を強化するには、チタニアがトレード・マークの支持面で活動的な段階との接触が重要です。それは、チタニアがアルミナ?...

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Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

エネルギー化学 3 D マップの取得フィルター透過電子顕微鏡トモグラフィー

本稿では、エネルギー フィルタ リング画像と電子線トモグラフィーを組み合わせた 3 D 化学マップを達成するためにプロトコルについて説明します。他のイメージング手法を区別することは困難である要素によって形成される 2 つの触媒担体の化学の分布を調べた。各アプリケーション重複元素それぞれ間隔がイオン化エッジのマッピングで構成されます。

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

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6.4K ビュー.  Université Claude Bernard Lyon 1. 本稿では、エネルギー フィルタ リング画像と電子線トモグラフィーを組み合わせた 3 D 化学マップを達成するためにプロトコルについて説明します。他のイメージング手法を区別することは困難である要素によって形成される 2 つの触媒担体の化学の分布を調べた。各アプリケーション重複元素それぞれ間隔がイオン化エッジのマッピングで構成されま?...

ビデオ: Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

個々のナノ粒子の3次元元素マッピングのためのエネルギー分散型X線トモグラフィー

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

工学

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

ダイナミックな相互作用の3次元構造解析のための相関顕微鏡

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

生物工学

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoautoの使用：ハイスループット自動クライオ電子断層撮影のためのプロトコル

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

電子クラ​​イオトモグラフィーによるミトコンドリアにおけるATP合成酵素二量体の可視化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

生物学

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

走査型透過電子トモグラフィーにより太い生物学的サンプルの調製と観察

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.

Here, we present a protocol to place simplified volumetric models into noisy, complex, tomographic 3D volumes. This allows the fast segmentation of actin filament densities, detection of systematic filament bending and of gaps in hair bundle filaments, as well as convenient quantification of volumetric model properties, such as distances.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. ...

神経科学

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

クライオ軟X線断層撮影による細胞の3D地図作成記述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

電子断層撮影による超構造保存クロマチン中の複製ドメインのイメージング

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

クライオ電子線トモグラフィー遠隔データ収集とサブトモグラム平均化

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

エネルギー化学 3 D マップの取得フィルター透過電子顕微鏡トモグラフィー

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個々のナノ粒子の3次元元素マッピングのためのエネルギー分散型X線トモグラフィー

ダイナミックな相互作用の3次元構造解析のための相関顕微鏡

Tomoautoの使用：ハイスループット自動クライオ電子断層撮影のためのプロトコル

電子クライオトモグラフィーによるミトコンドリアにおけるATP合成酵素二量体の可視化

走査型透過電子トモグラフィーにより太い生物学的サンプルの調製と観察

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

クライオ軟X線断層撮影による細胞の3D地図作成記述

電子断層撮影による超構造保存クロマチン中の複製ドメインのイメージング

クライオ電子線トモグラフィー遠隔データ収集とサブトモグラム平均化

クライオSTEMトモグラフィーによるオルガネラのin situ 可視化

エネルギー化学 3 D マップの取得フィルター透過電子顕微鏡トモグラフィー

要約

さらに動画を探す

この動画の章

個々のナノ粒子の3次元元素マッピングのためのエネルギー分散型X線トモグラフィー

ダイナミックな相互作用の3次元構造解析のための相関顕微鏡

Tomoautoの使用：ハイスループット自動クライオ電子断層撮影のためのプロトコル

電子クラ​​イオトモグラフィーによるミトコンドリアにおけるATP合成酵素二量体の可視化

走査型透過電子トモグラフィーにより太い生物学的サンプルの調製と観察

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

クライオ軟X線断層撮影による細胞の3D地図作成記述

電子断層撮影による超構造保存クロマチン中の複製ドメインのイメージング

クライオ電子線トモグラフィー遠隔データ収集とサブトモグラム平均化

クライオSTEMトモグラフィーによるオルガネラのin situ 可視化

電子クライオトモグラフィーによるミトコンドリアにおけるATP合成酵素二量体の可視化