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

Les microscopes optiques utilisent la lumière visible pour éclairer un objet, et leurs lentilles peuvent agrandir une image jusqu’à 2000 fois. Au lieu de la lumière, les microscopes électroniques utilisent un faisceau d’électrons avec une longueur d’onde très courte qui permet de grossir les objets de 2 à 50 millions de fois.
Un filament émetteur d’électrons génère le faisceau d’électrons, et des lentilles électrostatiques et électromagnétiques focalisent le faisceau sur un échantillon. L’environnement interne d’un microscope électronique est maintenu sous vide car les molécules d’air peuvent dévier le faisceau d’électrons.
Selon la façon dont le faisceau d’électrons interagit avec l’échantillon, les microscopes électroniques peuvent être divisés en deux types : le microscope électronique à transmission, ou MET, et le microscope électronique à balayage, ou MEB.
Dans le cadre de la MET, les électrons sont transmis à travers un échantillon mince, puis utilisés pour générer une image 2D. Lorsque le faisceau d’électrons traverse l’échantillon, des informations sur sa composition interne et sa structure peuvent être obtenues à l’aide de la MET.
Dans le MEB, le faisceau d'électrons balaie la surface d'un échantillon, puis les électrons réfléchis sont utilisés pour générer des informations sur la topographie 3D et la composition de la surface.

présentation de la microscopie Électronique

Les longueurs d'onde de la lumière visible limitent en fin de compte la résolution théorique maximale des images créées par les microscopes optiques. La plupart des microscopes optiques ne peuvent grossir que 1 000X, et quelques-uns peuvent grossir jusqu'à 1 500X. Les électrons, comme le rayonnement électromagnétique, peuvent se comporter comme des ondes, mais avec des longueurs d'onde de 0,005 nm, ils produisent une résolution nettement supérieure jusqu'à 0,05 nm, contre 500 nm pour la lumière visible. Un microscope électronique (ME) peut créer une image nette agrandie jusqu'à 2 000 000X. Ainsi, les MEs peuvent résoudre les structures subcellulaires et certaines structures moléculaires (par exemple, des brins simples d’ADN).

Il existe deux types fondamentaux d'ME : le microscope électronique à transmission (MET) et le microscope électronique à balayage (MEB). Le MET est quelque peu analogue au microscope optique à fond clair en termes de fonctionnement. Cependant, il utilise un faisceau d’électrons focalisé sur l’échantillon par le haut à l’aide d’une lentille magnétique (plutôt qu’une lentille en verre) et projeté à travers l’échantillon sur un détecteur. Les électrons traversent l’échantillon, puis le détecteur capture l’image.

Pour que les électrons traversent l'échantillon dans un MRT, l'échantillon doit être extrêmement fin (20 à 100 nm d'épaisseur). L'image est produite en raison de l'opacité variable dans diverses parties de l'échantillon. Cette opacité peut être renforcée en colorant l'échantillon avec des matériaux tels que des métaux lourds, qui sont denses aux électrons. MET nécessite que le faisceau et l’échantillon soient sous vide et que l’échantillon soit ultra fin et déshydraté.

Les MEBs forment des images de surfaces de spécimens, généralement à partir d'électrons qui sont expulsés de l'échantillon par un faisceau d'électrons. Cela peut créer des images très détaillées avec une apparence tridimensionnelle affichée sur un moniteur. En règle générale, les échantillons sont séchés et préparés avec des fixateurs qui réduisent les artefacts avant d'être recouverts par pulvérisation cathodique d'une fine couche de métal tel que l'or. Alors que la microscopie électronique à transmission nécessite des coupes très fines et permet de voir des structures internes telles que des organites et l'intérieur des membranes, la microscopie électronique à balayage peut être utilisée pour visualiser les surfaces d'objets plus grands (comme un grain de pollen) ainsi que les surfaces de de très petits échantillons.

Ce texte est adapté d'Openstax, Microbiologie, <a href="https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy">Section 2.3: Instruments of Microscopy</a>

Présentation de la Microscopie Électronique

Les longueurs d’onde de la lumière visible limitent finalement la résolution théorique maximale des images créées par les microscopes optiques. La plupart des microscopes optiques peuvent ...

Education

Enseignement

JoVE Core

Cell Biology

Biologie cellulaire

Unit 1

Unité 1

Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

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

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immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

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confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

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Fluorescent Methods for Protein Movement

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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 Biologie cellulaire illustre les processus cellulaires complexes dans des leçons vidéos complètes mais faciles à comprendre. Cette collection se concentre sur les composants et fonctions essentiels de la cellule ; cela inclut les organelles et les membranes ainsi que le transport et le métabolisme énergétique cellulaire. Elle est complétée par nos vidéos de scientifiques à l'action qui démontrent l'application de la biologie cellulaire dans des expériences concrètes.

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Electron Microscopy: TEM vs SEM

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