Name: 3d imaging with electron microscopy
Uploaded: 2023-04-30T12:01:40.000Z
Duration: 1 min 7 s
Channel: JoVE

生体試料の3D構造は、電子線トモグラフィーと単一粒子再構成の2つのクライオ電子顕微鏡技術を用いて決定することができます。
電子線トモグラフィーでは、サンプルは電子ビームに対して徐々に傾き、さまざまな角度から一連の2D画像をキャプチャします。次に、これらの2D画像をコンピュータソフトウェアを使用して積み重ね、サンプルの3D画像または断層像を生成します。
電子線トモグラフィーは、サンプルのin vivo構造的完全性を維持し、主に細胞、細胞小器官、高分子の天然環境での3D可視化に使用されます。
1粒子再構成では、精製されたタンパク質サンプルなどの単離されたin vitroサンプルがグリッド上に均一に分布します。
ランダムな向きに横たわっている個々のタンパク質分子の2D画像がキャプチャされます。次に、何千ものそのような画像が平均化され、位置合わせされて、サンプルの3D画像が再構築されます。

電子顕微鏡トモグラフィーと単一粒子再構成

Electron Microscope Tomography and Single-particle Reconstruction

透過型電子顕微鏡 (TEM) は、電子顕微鏡断層撮影や単一粒子再構成などの技術を活用して、生体サンプルの 3D 構造を決定するために使用できます。 単一粒子再構成では高分子および高分子複合体を in vitro 条件でのみ検査できますが、トモグラフィーではインビボでの細胞成分または小細胞の研究が可能です。

電子断層撮影法

電子断層撮影は、TEM または STEM (走査透過型電子顕微鏡) モードで実行できます。 STEM は、主に厚い生体標本の断層像を取得するために使用される技術です。 これは、走査型電子顕微鏡のサンプル表面走査方法を組み合わせたもので、電子ビームがサンプルに当たる各点で透過または散乱した電子が一連の検出器によって収集されます。

従来の電子断層撮影技術のバリエーションとして 2 軸断層撮影があります。この場合、サンプルは電子ビームに対して 2 つの軸の周りに傾けられます。 これにより、2 つの別個の断層像が得られ、これらの断層像が位置合わせされてサンプルの 3D 画像が生成されます。 二軸断層撮影法には、単軸断層撮影法に比べて、サンプルの拡張された特徴のより適切な再構成と、サンプルの深さの解像度の向上という 2 つの利点があります。

もう 1 つのバリエーションは電子クライオ断層撮影法です。この方法では、固定と脱水によって生物学的構造が損傷する可能性があるため、サンプルが極低温条件下で画像化されます。 厚いサンプルは電子ビームを遮断するため、この方法は主に厚さ 500 nm 未満の薄いサンプルに使用されます。 したがって、この技術は、精製された高分子複合体、ウイルス、または細菌細胞などの小細胞に限定されてきました。

単一粒子の再構築

単粒子分析は通常、原子に近い分解能で 3D 構造を生成するために極低温電子顕微鏡で使用されます。 これは、結晶化が難しい大きなまたは動的高分子複合体に最も適しており、X 線結晶構造解析の代替となります。 結晶化可能な複合体の場合、高解像度の構造を取得するには、単一粒子分析が最適な方法です。 この技術は、膜タンパク質、タンパク質複合体、クロマチン構造、リボソームやプロテアソームなどの高分子機構の研究に使用されています。

透過型電子顕微鏡(TEM)は、電子顕微鏡などの技術の助けを借りて、生物学的サンプルの3D構造を決定するために使用できます...

electron-microscope-tomography-and-single-particle-reconstruction

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Visualizing Cells, Tissues, and Molecules

細胞・組織・分子の可視化

KEY TERMS AND CONCEPTS

主要用語と概念

3d imaging with electron microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only.

Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It combines sample scanning with detectors that collect electrons transmitted or scattered where the beam hits the sample.

A common variation is dual-axis tomography. In this method, the sample is tilted around two axes relative to the electron beam. The two tomograms are then aligned to form a 3D image. Dual-axis tomography can improve the reconstruction of expanded features and increase depth resolution compared with single-axis tomography.

Another form is electron cryo-tomography. Samples are imaged under cryogenic conditions because fixation and dehydration can damage biological structures. This method is mainly used for thin samples, usually less than 500 nm thick, because thicker samples block the electron beam. It has therefore been limited to purified macromolecular complexes, viruses, and small cells such as bacterial cells.

Single-particle analysis is usually paired with cryo-electron microscopy to produce 3D structures with near-atomic resolution. It works best for large or dynamic macromolecular complexes that are hard to crystallize, so it can serve as an alternative to X-ray crystallography. When complexes can be crystallized, single-particle analysis is still a method of choice for obtaining high-resolution structures. It has been used to study membrane proteins, protein complexes, chromatin structure, and macromolecular machines such as ribosomes and proteasomes.

3D Imaging with Electron Microscopy

imaging biological samples with optical microscopy

imaging-biological-samples-with-optical-microscopy

How Light Microscopes Bring Samples Into View

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

phase contrast and differential interference contrast microscopy

phase-contrast-and-differential-interference-contrast-microscopy

Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

fixation and sectioning

Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

protein dynamics in living cells

Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

total-internal-reflection-fluorescence-microscopy

Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

super-resolution fluorescence microscopy

Sharper Cell Images with Fluorescence Microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

immunogold electron microscopy

Immunogold Labeling in Electron Microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

key terms and concepts

Learn how TEM creates 3D biological images using tomography and single-particle reconstruction, including STEM, dual-axis, and cryo-tomography methods.

3D Imaging with Electron Microscopy | JoVE K12

cell biology

JoVE Core 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

3D Imaging with Electron Microscopy

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