Name: electron microscopy sample prep methods
Uploaded: 2023-04-30T12:01:16.000Z
Duration: 1 min 20 s
Channel: JoVE

Les échantillons biologiques doivent être préparés pour la microscopie électronique afin de protéger l’échantillon des dommages causés par le puissant faisceau d’électrons et la basse pression créée par le vide.
La préparation des échantillons commence par la fixation chimique et la déshydratation complète. Les fixateurs chimiques tels que le glutaraldéhyde ou le formaldéhyde réticulent les protéines, tandis que le tétroxyde d’osmium est souvent utilisé pour fixer les lipides membranaires.
Ensuite, l’échantillon est déshydraté progressivement avec des concentrations croissantes d’éthanol ou d’acétone pour éviter le rétrécissement de l’échantillon.
Pour la MET, les échantillons sont noyés dans une résine époxy dure. Un ultramicrotome découpe ensuite l’échantillon en sections efficaces ultrafines à travers lesquelles les électrons peuvent passer.
Ces sections sont tachées de métaux lourds, tels que l’acétate d’uranyle. Les molécules qui lient les métaux apparaissent plus foncées et contrastent avec l’image.
Pour le MEB, les échantillons sont ensuite séchés à l’aide de dioxyde de carbone liquide dans un processus appelé séchage au point critique. Les échantillons sont ensuite recouverts d’une fine couche de métal conducteur, tel que de l’or, ce qui permet d’éviter les dommages thermiques à l’échantillon et d’offrir une meilleure qualité d’image.

préparation des Échantillons pour la microscopie Électronique

Preparation of Samples for Electron Microscopy

Pour être visualisés au microscope électronique, à transmission ou à balayage, les échantillons biologiques doivent être fixés (stabilisés) pour que le faisceau électronique ne les détruise pas et soigneusement séchés (desséchés/déshydratés) pour que le vide ne les affecte pas. La fixation doit être effectuée le plus rapidement possible car les propriétés de l'échantillon commenceront à changer dès qu'il sera retiré de son environnement naturel. Par exemple, dans un échantillon de tissu, les niveaux d’oxygène commencent à diminuer, provoquant une altération de l’apparence des mitochondries.

Avancées dans la Préparation des Échantillons TEM

Cryofixation

Dans cette technique, les petites cellules et organites peuvent être congelés par congélation en plongée, tandis que les cellules et tissus plus gros peuvent être congelés à haute pression. La congélation en plongée est obtenue en plaçant une petite quantité d'échantillon sur une grille ME froide puis en plongeant rapidement dans de l'éthane liquide rempli dans un réservoir et entouré d'azote liquide. La congélation rapide garantit que l'eau ne cristallise pas. Lors de la congélation à haute pression, de l'azote liquide est utilisé pour congeler rapidement l'échantillon et il est soumis à une pression d'environ 2 000 bars pour ralentir la formation de cristaux de glace. Les échantillons cryofixés sont sectionnés dans un cryo-ultramicrotome constitué d'un couteau en diamant congelé.

Projection D'ombre

La plate-forme rotative dans une chambre à vide contient l'échantillon, qui est ensuite recouvert de métal lourd. La rotation de l’échantillon assure un revêtement métallique sur la face avant et un effet d’ombre sur la face arrière. La technique est particulièrement utile lors de l’étude des détails ultrastructuraux de bactéries ou de virus, d’ADN, d’ARN ou de protéines isolées.

Coloration Négative

Les petits échantillons, tels que les ribosomes, les molécules d'enzymes, les virus, etc., peuvent être visualisés directement sans section. Les échantillons sont mélangés avec du molybdate d'ammonium, de l'acétate d'uranyle ou de l'acide phosphotungstique, qui sont opaques aux électrons. Une couche métallique se forme sur la grille, sauf là où se trouve l'échantillon, créant une image négative de l'échantillon. Le contraste est obtenu grâce au modèle de répartition des métaux.

Préparation D'échantillons Desséchés et Non Desséchés pour le MEB

MEB peut visualiser trois types d'échantillons, à savoir (i) desséchés et conducteurs, (ii) desséchés et non conducteurs, et (c) non desséchés (humides). Les échantillons naturellement conducteurs desséchés peuvent être facilement montés et imagés. Cependant, les échantillons desséchés et non conducteurs doivent être recouverts d'une couche conductrice pour protéger l'échantillon de la surchauffe et améliorer la qualité de l'image. Ces échantillons sont rendus conducteurs en recouvrant l'échantillon d'une fine couche d'or à l'aide d'une coucheuse par pulvérisation cathodique. Il utilise des ions d'argon pour éliminer les atomes d'or d'une plaque d'or et recouvrir la surface de l'échantillon d'or, rendant la surface conductrice des électrons.

Les échantillons humides peuvent dégazer dans l’environnement sous vide du microscope.

Le MEB humide, également appelé MEB environnemental ou MEBE, doit contrôler la sublimation de l'eau dans l'échantillon lors de l'imagerie. Cela se fait en enduisant d’abord l’échantillon d’une fine couche d’un polymère membranaire transparent aux faisceaux d’électrons et séparant l’échantillon humide du vide. Ensuite, la température diminue, ce qui diminue la pression de vapeur saturée. Ces conditions permettent le fonctionnement de l'instrument sous un vide plus élevé qui préserve les caractéristiques ultrastructurales de l'échantillon et réduit le bruit. Un Deben Coolstage peut également être utilisé pour imager des échantillons humides en les congelant à -25 °C, ce qui préserve la structure de l'échantillon et empêche le dégazage pendant que les électrons le bombardent.

Préparation des Échantillons pour la Microscopie Électronique

Pour être visualisés par un microscope électronique, qu’il s’agisse d’une transmission ou d’un balayage, les échantillons biologiques doivent être fixés (stabilisés) de sorte que le faisceau d’électrons ne ...

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 is why early stabilization matters.

TEM sample prep can use cryofixation, shadow casting, or negative staining. In cryofixation, small cells and organelles are plunge frozen on a cold EM grid in liquid ethane surrounded by liquid nitrogen. Larger cells and tissues can be frozen by high-pressure freezing with liquid nitrogen and about 2000 bar of pressure. This slows ice crystal formation. Cryofixed samples are then cut in a cryo-ultramicrotome with a frozen diamond knife.

Shadow casting places the sample on a rotating platform in a vacuum chamber and coats it with heavy metal. The rotation helps cover the front side and creates a shadow effect on the back side. This method is useful for ultrastructural details in bacteria, viruses, DNA, RNA, and isolated proteins. Negative staining is used for small samples such as ribosomes, enzyme molecules, and viruses. The sample is mixed with an electron-opaque stain such as ammonium molybdate, uranyl acetate, or phosphotungstic acid, which leaves a metal layer around the sample and forms a negative image.

SEM sample prep depends on whether the sample is desiccated, conductive, non-conductive, or wet. Naturally conductive dry samples can be mounted and imaged directly. Dry but non-conductive samples need a thin gold coating from a sputter coater. Argon ions knock gold atoms off a gold plate and deposit them on the sample surface. This makes the surface conductive and helps prevent overheating while improving image quality.

Wet samples can outgas in the vacuum of the microscope. For these specimens, wet SEM, also called Environmental SEM or ESEM, controls water sublimation during imaging. One approach uses a thin membranous polymer that is transparent to electron beams and separates the wet specimen from the vacuum. The temperature is then lowered, which reduces saturated vapor pressure. A Deben Coolstage can also image wet samples by freezing them to -25°C, which preserves structure and limits outgassing while electrons strike the specimen.

Electron Microscopy Sample Prep Methods

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 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

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Antibody Staining in Cells and Tissues

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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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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...

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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

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

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

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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 biological samples are fixed, dried, coated, or frozen for TEM and SEM, including cryofixation, negative staining, and wet sample imaging.

Electron Microscopy Sample Prep Methods | JoVE K12

cell biology

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Electron Microscopy Sample Prep Methods

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