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

Bir taramalı elektron mikroskobu veya SEM, numune yüzeyinin topografyası ve bileşimi hakkında bilgi sağlamak için bir numuneyi bir elektron demetiyle tarar. 
SEM, pozitif yüklü bir anot tarafından aşağı doğru çekilen negatif yüklü bir elektron ışını oluşturmak için bir elektron tabancası kullanır. Elektromanyetik lensler daha sonra elektron demetini yoğunlaştırır ve çapını azaltır.
Daha sonra, tarama bobinleri bu odaklanmış elektron demetini x ve y ekseni boyunca saptırır ve numune yüzeyinin dikdörtgen bir alanını taramasına izin verir.
Yüksek enerjili birincil elektron ışını, bir numunenin atomlarındaki elektronları uyararak düşük enerjili ikincil elektronların salınmasına yol açar. Bu ikincil elektronlar dedektör tarafından okunur ve numune yüzeyinin 3 boyutlu bir görüntüsünü oluşturmak için kullanılır. 
Elektron ışını-numune etkileşimi ayrıca, numunede bulunan demir ve nikel gibi farklı elementler hakkında bilgi sağlayan karakteristik X-ışınları üretir.

taramalı elektron mikroskobu

Scanning Electron Microscopy

Taramalı elektron mikroskobu (SEM), numune yüzeyini iki boyutlu bir şekilde tarayan bir elektron ışını kullanarak bir numunenin yüzey özelliklerini incelemek için kullanılır. Tipik olarak genişliği ~1 santimetre ila 5 mikrometre arasındaki alanlar görüntülenebilir. SEM bakterileri, virüsleri, dokuları ve böcekler gibi daha büyük örnekleri görüntülemek için kullanılabilir. Geleneksel SEM, 20X ila 30.000X arasında değişen bir büyütme ve 50 ila 100 nanometre arasında uzaysal çözünürlük sağlar. Temel prensipler Elektron tabancası tarafından salınan hızlandırılmış elektronlar yüksek kinetik enerjiye sahiptir (5-30 keV arasında değişir). Elektron-örnek etkileşimleri elektronların yavaşlamasına ve enerjinin farklı sinyaller şeklinde dağılmasına yol açar. Elektronlar iki tür saçılmaya maruz kalır: elastik ve elastik olmayan. Esnek olmayan saçılma ikincil elektronların emisyonuna neden olur. Bu düşük enerjili elektronlar (~50 eV), atomun yüzeyini terk etmeye yetecek kadar enerji elde eden örnek atomların dış kabuk elektronlarıdır. Numunenin iç bölgelerinden çıkan elektronların enerji seviyesi numune yüzeyinden çıkamayacak kadar düşük olduğundan, ikincil elektronların saçılmasıyla yalnızca topografik bilgi sağlanır. X ışınları aynı zamanda numunedeki atomların ayrı yörüngelerindeki (kabuklarındaki) elektronlarla gelen elektronların esnek olmayan çarpışmalarıyla da üretilir. Uyarılmış elektronlar daha düşük enerji durumlarına döndükçe, sabit dalga boyunda X-ışınları üretirler (belirli bir element için farklı kabuklardaki elektronların enerji seviyelerindeki farkla bağlantılı olarak). Böylece elektron ışınıyla uyarılan mineraldeki her element için karakteristik X-ışınları üretilir. Öte yandan elastik saçılma, örnek atomlardan çıkan elektronlardan kaynaklanmaz. Ana elektron demeti çekirdekle etkileşimden sonra geri saçılır. Bu elektronlar enerjilerini veya hızlarını değiştirmezler, çekirdekle etkileşimlerine göre yönlerini değiştirirler. Bu elektronların tespiti birleşik veri sağlar ve bunların farklı atom ağırlıklarına sahip atomlarla etkileşimi üzerine değişen kontrastları, kullanıcının numune bileşimindeki farklılıkları ayırt etmesine olanak tanır. Biyolojik numunelerde bu, altın veya demir gibi daha ağır atomik ağırlıklara sahip gömülü veya bağlı nanoparçacıkları ve nanoyapıları incelemek için kullanılabilir.

Taramalı Elektron Mikroskobu

Taramalı elektron mikroskobu (SEM), numune yüzeyini bir sıcaklıkta tarayan bir elektron ışını kullanarak bir numunenin yüzey özelliklerini incelemek için kullanılır.

Education

JoVE Core

Cell Biology

Hücre Biyolojisi

Unit 1

Ünite 1

Visualizing Cells, Tissues, and Molecules

Hücrelerin, Dokuların ve Moleküllerin Görülebilir Kılınması

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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

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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...

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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...

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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...

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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

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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...

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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...

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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...

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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...

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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...

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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

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How SEM Reveals Surface Details

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