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

Skaningowy mikroskop elektronowy lub SEM skanuje próbkę za pomocą wiązki elektronów, aby dostarczyć informacji o topografii i składzie powierzchni próbki. 
SEM wykorzystuje działo elektronowe do generowania ujemnie naładowanej wiązki elektronów przyciąganej w dół przez dodatnio naładowaną anodę. Soczewki elektromagnetyczne koncentrują wtedy wiązkę elektronów i zmniejszają jej średnicę.
Następnie cewki skanujące odchylają tę skupioną wiązkę elektronów wzdłuż osi x i y i pozwalają jej zeskanować prostokątny obszar powierzchni próbki.
Wysokoenergetyczna pierwotna wiązka elektronów wzbudza elektrony w atomach próbki, prowadząc do uwolnienia niskoenergetycznych elektronów wtórnych. Te wtórne elektrony są odczytywane przez detektor i wykorzystywane do generowania obrazu 3D powierzchni próbki. 
Oddziaływanie wiązka elektronów-próbka wytwarza również charakterystyczne promieniowanie rentgenowskie, które dostarcza informacji o różnych pierwiastkach, takich jak żelazo i nikiel, obecnych w próbce.

skaningowa mikroskopia elektronowa

Scanning Electron Microscopy

Skaningowy mikroskop elektronowy (SEM) służy do badania cech powierzchni próbki za pomocą wiązki elektronów, która skanuje powierzchnię próbki w sposób dwuwymiarowy. Zazwyczaj można obrazować obszary o szerokości od ~1 centymetra do 5 mikrometrów. SEM można wykorzystać do obrazowania bakterii, wirusów, tkanek, a także większych próbek, takich jak owady. Konwencjonalny SEM zapewnia powiększenie w zakresie od 20X do 30 000X i rozdzielczość przestrzenną od 50 do 100 nanometrów. Fundamentalne zasady Przyspieszone elektrony uwalniane przez działo elektronowe mają wysoką energię kinetyczną (w zakresie 5-30 keV). Interakcje elektron-próbka prowadzą do spowolnienia elektronów i rozproszenia energii w postaci różnych sygnałów. Elektrony ulegają dwóm rodzajom rozpraszania: elastycznemu i niesprężystemu. Rozpraszanie nieelastyczne powoduje emisję elektronów wtórnych. Te niskoenergetyczne elektrony (~50 eV) to elektrony z zewnętrznej powłoki atomów próbki, które uzyskują energię wystarczającą do opuszczenia powierzchni atomu. Rozpraszanie elektronów wtórnych dostarcza jedynie informacji topograficznych, ponieważ poziom energii elektronów opuszczających wewnętrzne obszary próbki jest zbyt niski, aby opuścić powierzchnię próbki. Promienie rentgenowskie powstają także w wyniku niesprężystych zderzeń padających elektronów z elektronami na dyskretnych orbitali (powłokach) atomów w próbce. Gdy wzbudzone elektrony powracają do niższych stanów energetycznych, emitują promieniowanie rentgenowskie o stałej długości fali (związanej z różnicą poziomów energii elektronów na różnych powłokach danego pierwiastka). W ten sposób dla każdego pierwiastka minerału wzbudzonego wiązką elektronów wytwarzane jest charakterystyczne promieniowanie rentgenowskie. Z drugiej strony, rozpraszanie elastyczne nie jest spowodowane wyparciem elektronów z atomów próbki. Główna wiązka elektronów ulega rozproszeniu wstecznemu po interakcji z jądrem. Elektrony te nie zmieniają swojej energii ani prędkości, ale zmieniają kierunek w oparciu o interakcję z jądrem. Wykrywanie tych elektronów dostarcza informacji o składzie, a ich zmienny kontrast w wyniku interakcji z atomami o różnych masach atomowych pozwala użytkownikowi rozróżnić różnice w składzie próbki. W próbkach biologicznych można to wykorzystać do badania osadzonych lub przyłączonych nanocząstek i nanostruktur o większej masie atomowej, takich jak złoto lub żelazo.

Skaningowa mikroskopia elektronowa

Skaningowy mikroskop elektronowy (SEM) służy do badania cech powierzchni próbki za pomocą wiązki elektronów, która skanuje powierzchnię próbki w ...

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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

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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

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Confocal Microscopy for Sharp Cell Images

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total internal reflection fluorescence microscopy

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Surface-Only Imaging with TIRF Microscopy

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Atomic Force Microscopy for Surface Mapping

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Sharper Cell Images with Fluorescence Microscopy

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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

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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