Name: electron microscopy: tem vs sem
Uploaded: 2023-04-30T12:00:52.000Z
Duration: 1 min 25 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

Overview of Electron Microscopy

Lichtmikroskope verwenden sichtbares Licht, um ein Objekt zu beleuchten, und ihre Linsen können ein Bild bis zu 2000-fach vergrößern. Anstelle von Licht verwenden Elektronenmikroskope einen Elektronenstrahl mit einer sehr kurzen Wellenlänge, der die Vergrößerung von Objekten um das 2- bis 50-millionenfache ermöglicht.
Ein elektronenemittierender Glühfaden erzeugt den Elektronenstrahl, und elektrostatische und elektromagnetische Linsen fokussieren den Strahl auf eine Probe. Die innere Umgebung eines Elektronenmikroskops wird unter Vakuum gehalten, da Luftmoleküle den Elektronenstrahl ablenken können.
Je nachdem, wie der Elektronenstrahl mit der Probe interagiert, können Elektronenmikroskope in zwei Typen unterteilt werden – das Transmissionselektronenmikroskop (TEM) und das Rasterelektronenmikroskop (REM).
Bei der TEM werden die Elektronen durch eine dünne Probe geleitet und anschließend zur Erzeugung eines 2D-Bildes verwendet. Während der Elektronenstrahl die Probe durchdringt, können mit Hilfe von TEM Informationen über ihre innere Zusammensetzung und Struktur gewonnen werden.
Beim REM tastet der Elektronenstrahl die Oberfläche einer Probe ab, und dann werden die reflektierten Elektronen verwendet, um Informationen über die 3D-Topographie und die Zusammensetzung der Oberfläche zu erzeugen.

Überblick über die elektronenmikroskopie

Die Wellenlängen des sichtbaren Lichts begrenzen letztlich die maximale theoretische Auflösung von Bildern, die Lichtmikroskope erzeugen. Die meisten Lichtmikroskope können nur 1000-fach vergrößern, einige sogar bis zu 1500-fach. Elektronen können sich wie elektromagnetische Strahlung wie Wellen verhalten, erzeugen aber bei Wellenlängen von 0,005 nm eine deutlich höhere Auflösung von bis zu 0,05 nm im Vergleich zu 500 nm für sichtbares Licht. Ein Elektronenmikroskop (EM) kann ein scharfes Bild erzeugen, das bis zu 2.000.000-fach vergrößert wird. Somit können EMs subzelluläre Strukturen und einige molekulare Strukturen (z. B. einzelne DNA-Stränge) auflösen.

Es gibt zwei Grundtypen von EM – das Transmissionselektronenmikroskop (TEM) und das Rasterelektronenmikroskop (REM). Das TEM ähnelt in seiner Funktionsweise in gewisser Weise dem Hellfeld-Lichtmikroskop. Dabei wird jedoch ein Elektronenstrahl verwendet, der mithilfe einer magnetischen Linse (anstelle einer Glaslinse) von oben auf die Probe fokussiert und durch die Probe auf einen Detektor projiziert wird. Elektronen passieren die Probe und dann erfasst der Detektor das Bild.

Damit Elektronen in einem TEM durch die Probe gelangen können, muss die Probe extrem dünn sein (20–100 nm dick). Das Bild entsteht aufgrund der unterschiedlichen Opazität in verschiedenen Teilen der Probe. Diese Opazität kann durch Anfärben der Probe mit Materialien wie Schwermetallen, die elektronendicht sind, verstärkt werden. TEM erfordert, dass sich Strahl und Probe im Vakuum befinden und dass die Probe ultradünn und dehydriert ist.

REMs erzeugen Bilder von Probenoberflächen, üblicherweise anhand von Elektronen, die durch einen Elektronenstrahl von der Probe abgeschossen werden. Dadurch können hochdetaillierte Bilder mit dreidimensionalem Erscheinungsbild erstellt werden, die auf einem Monitor angezeigt werden. Typischerweise werden die Proben getrocknet und mit Fixiermitteln präpariert, die Artefakte reduzieren, bevor sie mit einer dünnen Metallschicht, beispielsweise Gold, durch Sputtern beschichtet werden. Während die Transmissionselektronenmikroskopie sehr dünne Schnitte erfordert und es ermöglicht, innere Strukturen wie Organellen und das Innere von Membranen zu sehen, kann die Rasterelektronenmikroskopie verwendet werden, um die Oberflächen größerer Objekte (z. B. eines Pollenkörnchens) sowie die Oberflächen von zu betrachten sehr kleine Proben.

Dieser Text ist adaptiert von Openstax, Microbiology, <a href="https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy">Section 2.3: Instruments of Microscopy</a>

Überblick über die Elektronenmikroskopie

Die Wellenlängen des sichtbaren Lichts begrenzen letztlich die maximale theoretische Auflösung der mit Lichtmikroskopen erzeugten Bilder. Die meisten Lichtmikroskope können ...

Education

Lehre

JoVE Core

Cell Biology

Zellbiologie

Unit 1

Einheit 1

Visualizing Cells, Tissues, and Molecules

Visualisierung von Zellen, Geweben und Molekülen

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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 strands of DNA.

There are two main types of electron microscopes: the transmission electron microscope, or TEM, and the scanning electron microscope, or SEM. A TEM works in a way that is somewhat similar to a brightfield light microscope. It uses an electron beam focused from above with a magnetic lens, not a glass lens, and sends the beam through the sample to a detector. The detector captures the image after electrons pass through the specimen.

A TEM sample must be extremely thin, usually 20 to 100 nm thick, so electrons can pass through it. Different parts of the specimen block electrons by different amounts, which creates the image. Staining with electron-dense materials such as heavy metals can increase this contrast. TEM also requires a vacuum, and the sample must be ultrathin and dehydrated.

SEM is used to view the surface of a specimen. It forms images from electrons that are knocked off the sample by an electron beam. This produces very detailed images with a three-dimensional appearance on a monitor. Specimens are usually dried and treated with fixatives to reduce artifacts before they are sputter-coated with a thin layer of metal, such as gold. TEM is best for internal structures such as organelles and membrane interiors, while SEM is useful for surfaces, including larger objects like a pollen grain, as well as very small samples.

Electron Microscopy: TEM vs SEM

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 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

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 electron microscopy uses electrons for high-resolution images, and compare TEM and SEM for viewing internal structures and surface detail.

Electron Microscopy: TEM vs SEM | JoVE K12

cell biology

JoVE Core Zellbiologie veranschaulicht komplexe zelluläre Prozesse in leicht verständlichen und dennoch umfassenden animierten Videolektionen. Diese Sammlung konzentriert sich auf die wesentlichen Bestandteile und Funktionen von Zellen, einschließlich Organellen und Membranen, Zelltransport und Zellenergie. Ergänzt wird sie durch Videos von Wissenschaftlern in Aktion, die die Anwendung der Zellbiologie in realen Forschungsexperimenten demonstrieren.

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SC.912.L.14.3

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Electron Microscopy: TEM vs SEM

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