Name: how sem reveals surface details
Uploaded: 2023-04-30T12:01:00.000Z
Duration: 1 min 7 s
Channel: JoVE

走査型電子顕微鏡またはSEMは、電子ビームでサンプルをスキャンして、サンプル表面のトポグラフィーと組成に関する情報を提供します。 
SEMは、電子銃を使用して、正に帯電した陽極によって下向きに引き付けられた負に帯電した電子ビームを生成します。次に、電磁レンズが電子ビームを集中させ、その直径を小さくします。
次に、走査コイルは、この集束電子ビームをx軸とy軸に沿って偏向させ、サンプル表面の長方形の領域をスキャンできるようにします。
高エネルギーの一次電子ビームは、試料の原子内の電子を励起し、低エネルギーの二次電子を放出します。これらの二次電子は検出器によって読み取られ、サンプル表面の3D画像を生成するために使用されます。 
電子ビームとサンプルの相互作用により、サンプルに存在する鉄やニッケルなどのさまざまな元素に関する情報を提供する特徴的なX線も生成されます。

走査型電子顕微鏡で観察

Scanning Electron Microscopy

走査型電子顕微鏡 (SEM) は、サンプル表面を 2 次元で走査する電子ビームを使用してサンプルの表面特徴を研究するために使用されます。 通常、幅約 1 センチメートルから 5 マイクロメートルまでの領域を画像化できます。 SEM は、細菌、ウイルス、組織だけでなく、昆虫などの大きなサンプルの画像化にも使用できます。 従来の SEM の倍率は 20 ～ 30,000 倍、空間分解能は 50 ～ 100 ナノメートルです。

基本原則

電子銃によって放出される加速電子は、高い運動エネルギー (5 ～ 30 keV の範囲) を持っています。 電子とサンプルの相互作用により、電子が減速し、さまざまな信号の形でエネルギーが散逸されます。 電子は、弾性散乱と非弾性散乱という 2 種類の散乱を受けます。 非弾性散乱により二次電子が放出されます。 これらの低エネルギー電子 (約 50 eV) はサンプル原子の外殻電子で、原子の表面から出るのにちょうど十分なエネルギーを獲得します。 サンプルの内部領域から出る電子のエネルギーレベルはサンプル表面から出るには低すぎるため、二次電子の散乱によってトポグラフィー情報のみが提供されます。

X 線は、入射電子とサンプル内の原子の離散軌道 (シェル) 内の電子との非弾性衝突によっても生成されます。 励起された電子がより低いエネルギー状態に戻ると、固定波長の X 線が生成されます (特定の元素の異なるシェル内の電子のエネルギー レベルの違いに関連します)。 したがって、電子線によって励起された鉱物の各元素に対して特性 X 線が発生します。

一方、弾性散乱は、サンプル原子から放出された電子によって引き起こされるものではありません。 電子の主ビームは原子核との相互作用後に後方散乱されます。 これらの電子はエネルギーや速度を変えませんが、原子核との相互作用に基づいて方向を変えます。 これらの電子の検出により組成情報が得られ、異なる原子量の原子との相互作用によるコントラストの変化により、ユーザーはサンプル組成の違いを区別することができます。 生物学的サンプルでは、これを使用して、金や鉄などのより重い原子量をもつ、埋め込まれたり付着したナノ粒子やナノ構造を研究することができます。

走査型電子顕微鏡(SEM)は、サンプルの表面をスキャンする電子ビームを使用して、サンプルの表面の特徴を研究するために使用されます...

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

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 releases accelerated electrons. These electrons carry high kinetic energy, usually in the range of 5 to 30 keV. When they hit the sample, the electrons lose energy through electron-sample interactions.

These interactions produce different signals through elastic and inelastic scattering. Inelastic scattering creates secondary electrons, which are low-energy electrons of about 50 eV. They come from the outer shell electrons of sample atoms and can escape from the surface, so they mainly show topography, or surface shape.

Inelastic collisions can also generate X-rays. These X-rays come from electrons in discrete atomic shells that drop back to lower energy states. Because each element produces characteristic X-rays with a fixed wavelength, SEM can help identify elements in a mineral.

Elastic scattering gives a different result. In this case, the main beam of electrons is backscattered after interacting with the nucleus. These electrons keep the same energy and speed, but they change direction. Detecting backscattered electrons provides compositional information and can show contrast between atoms of different atomic weights.

This contrast is useful in biological samples as well. It can help researchers study embedded or attached nanoparticles and nanostructures with heavier atomic weights, such as gold or iron. That makes SEM valuable for both surface detail and composition.

How SEM Reveals Surface Details

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

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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

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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

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

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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

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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...

key terms and concepts

Learn how a scanning electron microscope scans samples, produces secondary electrons, X-rays, and backscattered signals, and reveals surface detail and composition.

How SEM Reveals Surface Details | 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。

How SEM Reveals Surface Details

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