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

주사 전자 현미경 또는 SEM은 전자 빔으로 샘플을 스캔하여 샘플 표면의 지형과 구성에 대한 정보를 제공합니다. 
SEM은 전자총을 사용하여 양전하를 띤 양극에 의해 아래쪽으로 끌어당기는 음전하를 띤 전자빔을 생성합니다. 그런 다음 전자기 렌즈는 전자빔을 집중시키고 직경을 줄입니다.
다음으로, 스캐닝 코일은 x축과 y축을 따라 이 집속된 전자빔을 편향시키고 샘플 표면의 직사각형 영역을 스캔할 수 있도록 합니다.
고에너지 1차 전자빔은 샘플의 원자에서 전자를 여기시켜 저에너지 2차 전자를 방출합니다. 이러한 2차 전자는 검출기에서 판독되어 샘플 표면의 3D 이미지를 생성하는 데 사용됩니다. 
또한 전자빔-시료 상호 작용은 시료에 존재하는 철 및 니켈과 같은 다양한 원소에 대한 정보를 제공하는 특징적인 X선을 생성합니다.

주사전자현미경

Scanning Electron Microscopy

주사전자현미경(SEM)은 시료 표면을 2차원 방식으로 스캔하는 전자빔을 사용하여 시료의 표면 특징을 연구하는 데 사용됩니다. 일반적으로 너비가 ~1cm~5μm 사이의 영역을 이미지화할 수 있습니다. SEM은 박테리아, 바이러스, 조직은 물론 곤충과 같은 더 큰 표본을 이미지화하는 데 사용할 수 있습니다. 기존 SEM은 20X~30,000X 범위의 배율과 50~100나노미터의 공간 분해능을 제공합니다.

기본 원리

전자총에 의해 방출된 가속된 전자는 높은 운동 에너지(5-30keV 범위)를 갖습니다. 전자-표본 상호 작용으로 인해 전자가 감속되고 에너지가 다른 신호 형태로 소산됩니다. 전자는 탄성 산란과 비탄성 산란이라는 두 가지 유형의 산란을 겪습니다. 비탄성 산란으로 인해 2차 전자가 방출됩니다. 이러한 저에너지 전자(~50eV)는 원자 표면을 떠나기에 충분한 에너지를 얻는 표본 원자의 외부 껍질 전자입니다. 시료의 내부 영역에서 나가는 전자의 에너지 준위가 시료 표면을 빠져나가기에는 너무 낮기 때문에 2차 전자의 산란에 의해 지형학적 정보만 제공됩니다.

X선은 또한 표본에 있는 원자의 개별 궤도(껍질)에 있는 전자와 입사 전자의 비탄성 충돌에 의해 생성됩니다. 여기된 전자가 더 낮은 에너지 상태로 돌아가면서 고정된 파장의 X선을 생성합니다(주어진 원소에 대해 서로 다른 껍질에 있는 전자의 에너지 수준 차이와 관련됨). 따라서 전자빔에 의해 여기된 광물의 각 원소에 대해 특성 X선이 생성됩니다.

반면에 탄성 산란은 표본 원자에서 전자가 이탈하여 발생하지 않습니다. 전자의 주요 빔은 핵과 상호작용한 후 후방 산란됩니다. 이 전자들은 에너지나 속도를 바꾸지 않고 핵과의 상호작용에 따라 방향을 바꿉니다. 이러한 전자를 감지하면 구성 정보가 제공되며, 서로 다른 원자량의 원자와의 상호 작용에 따른 다양한 대비를 통해 사용자는 표본 구성의 차이를 구별할 수 있습니다. 생물학적 표본에서 이는 금이나 철과 같이 더 무거운 원자량을 갖는 내장되거나 부착된 나노입자 및 나노구조를 연구하는 데 사용할 수 있습니다.

주사 전자 현미경 (SEM)은 샘플 표면을 스캔하는 전자빔을 사용하여 샘플의 표면 특징을 연구하는 데 사용됩니다.

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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

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

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

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

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 Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

How SEM Reveals Surface Details

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