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

Biologische Proben müssen für die Elektronenmikroskopie vorbereitet werden, um die Probe vor Beschädigungen durch den starken Elektronenstrahl und den durch das Vakuum erzeugten niedrigen Druck zu schützen.
Die Probenvorbereitung beginnt mit der chemischen Fixierung und der vollständigen Dehydratisierung. Chemische Fixiermittel wie Glutaraldehyd oder Formaldehyd vernetzen die Proteine, während Osmiumtetroxid häufig zur Fixierung der Membranlipide verwendet wird.
Anschließend wird die Probe schrittweise mit steigenden Konzentrationen von Ethanol oder Aceton dehydriert, um ein Schrumpfen der Probe zu vermeiden.
Für TEM werden die Proben in ein hartes Epoxidharz eingebettet. Ein Ultramikrotom schneidet die Probe dann in ultradünne Querschnitte, die Elektronen passieren können.
Diese Abschnitte sind mit Schwermetallen wie Uranylacetat gefärbt. Moleküle, die die Metalle binden, erscheinen dunkler und sorgen für Kontrast zum Bild.
Für das REM werden die Proben mit flüssigem Kohlendioxid in einem Prozess weiter getrocknet, der als Critical-Point-Trocknung bezeichnet wird. Die Proben werden dann mit einer dünnen Schicht aus leitfähigem Metall, wie z. B. Gold, gesputtert, um eine thermische Schädigung der Probe zu verhindern und eine bessere Bildqualität zu erzielen.

vorbereitung von proben für die elektronenmikroskopie

Preparation of Samples for Electron Microscopy

Um mit einem Transmissions- oder Rasterelektronenmikroskop sichtbar gemacht zu werden, müssen biologische Proben fixiert (stabilisiert) werden, damit der Elektronenstrahl sie nicht zerstört, und gründlich getrocknet (ausgetrocknet/dehydriert) werden, damit das Vakuum sie nicht beeinträchtigt. Die Fixierung muss so schnell wie möglich erfolgen, da sich die Eigenschaften der Probe verändern, sobald sie aus ihrer natürlichen Umgebung entfernt wird. Beispielsweise beginnt in einer Gewebeprobe der Sauerstoffgehalt zu sinken, was zu einem veränderten Erscheinungsbild der Mitochondrien führt.

Fortschritte in der TEM-Probenvorbereitung

Kryofixierung

Bei dieser Technik können kleine Zellen und Organellen durch Tauchgefrieren eingefroren werden, während größere Zellen und Gewebe durch Hochdruck eingefroren werden können. Das Tauchgefrieren wird erreicht, indem eine kleine Probenmenge auf ein kaltes EM-Gitter gelegt und dann schnell in flüssiges Ethan getaucht wird, das in einem Reservoir gefüllt und von flüssigem Stickstoff umgeben ist. Das schnelle Gefrieren sorgt dafür, dass das Wasser nicht kristallisiert. Beim Hochdruckgefrieren wird flüssiger Stickstoff verwendet, um die Probe schnell einzufrieren, und sie wird einem Druck von etwa 2000 bar ausgesetzt, um die Eiskristallbildung zu verlangsamen. Kryofixierte Proben werden in einem Kryo-Ultramikrotom geschnitten, das aus einem gefrorenen Diamantmesser besteht.

Schattenwurf

Die rotierende Plattform in einer Vakuumkammer hält die Probe, die dann mit Schwermetall beschichtet wird. Die Rotation der Probe sorgt für eine Metallbeschichtung auf der Vorderseite und einen Schatteneffekt auf der Rückseite. Die Technik ist besonders nützlich, wenn ultrastrukturelle Details von Bakterien oder Viren, DNA, RNA oder isolierten Proteinen untersucht werden.

Negative Färbung

Kleine Proben wie Ribosomen, Enzymmoleküle, Viren usw. können ohne Schnitte direkt abgebildet werden. Die Proben werden mit Ammoniummolybdat, Uranylacetat oder Phosphorwolframsäure gemischt, die elektronenundurchlässig sind. Auf dem Gitter wird eine Metallschicht gebildet, außer dort, wo die Probe liegt, wodurch ein Negativbild der Probe entsteht. Der Kontrast wird durch das Muster der Metallverteilung erreicht.

Vorbereiten getrockneter und nicht getrockneter Proben für die REM

SEM kann drei Arten von Proben abbilden, nämlich (i) ausgetrocknet und leitend, (ii) ausgetrocknet und nicht leitend und (c) nicht ausgetrocknet (nass). Natürlich leitfähige Proben, die getrocknet sind, können einfach montiert und abgebildet werden. Allerdings müssen ausgetrocknete und nicht leitende Proben mit einer leitenden Schicht überzogen werden, um die Probe vor Überhitzung zu schützen und die Bildqualität zu verbessern. Diese Proben werden leitfähig gemacht, indem die Probe mithilfe eines Sputtercoaters mit einer dünnen Goldschicht beschichtet wird. Es nutzt Argonionen, um Goldatome von einer Goldplatte abzustoßen und die Probenoberfläche mit Gold zu überziehen, wodurch die Oberfläche für Elektronen leitend wird.

Nasse Proben können in der Vakuumumgebung des Mikroskops ausgasen.

Nass-REM, auch Umwelt-REM oder ESEM genannt, muss die Wassersublimation in der Probe während der Bildgebung kontrollieren. Dazu wird die Probe zunächst mit einer dünnen Schicht eines membranösen Polymers beschichtet, das für Elektronenstrahlen transparent ist und die nasse Probe vom Vakuum trennt. Dann wird die Temperatur gesenkt, wodurch der Sättigungsdampfdruck abnimmt. Diese Bedingungen ermöglichen den Betrieb des Instruments bei einem höheren Vakuum, wodurch die ultrastrukturellen Eigenschaften der Probe erhalten bleiben und das Rauschen reduziert wird. Ein Deben Coolstage kann auch zur Abbildung nasser Proben verwendet werden, indem diese auf –25 °C eingefroren werden, wodurch die Probenstruktur erhalten bleibt und ein Ausgasen verhindert wird, während sie mit Elektronen bombardiert wird.

Vorbereitung von Proben für die Elektronenmikroskopie

Um mit einem Elektronenmikroskop sichtbar gemacht zu werden, entweder durch Transmission oder Abtastung, müssen biologische Proben fixiert (stabilisiert) werden, damit der Elektronenstrahl ...

preparation-of-samples-for-electron-microscopy

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 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

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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...

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Fluorescent Antibody Staining in Cells

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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

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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...

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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...

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3D Imaging with Electron Microscopy

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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

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.

Electron Microscopy Sample Prep Methods

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