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

Las muestras biológicas deben prepararse para microscopía electrónica para proteger la muestra del daño causado por el potente haz de electrones y la baja presión creada por el vacío.
La preparación de la muestra comienza con la fijación química y la deshidratación completa. Los fijadores químicos, como el glutaraldehído o el formaldehído, entrecruzan las proteínas, mientras que el tetróxido de osmio se utiliza a menudo para fijar los lípidos de la membrana.
A continuación, la muestra se deshidrata gradualmente con concentraciones crecientes de etanol o acetona para evitar la contracción de la muestra.
Para TEM, las muestras se incrustan en una resina epoxi dura. A continuación, un ultramicrótomo corta la muestra en secciones transversales ultrafinas a través de las cuales pueden pasar los electrones.
Estas secciones están teñidas con metales pesados, como el acetato de uranilo. Las moléculas que se unen a los metales parecen más oscuras y proporcionan contraste a la imagen.
Para SEM, las muestras se secan aún más utilizando dióxido de carbono líquido en un proceso llamado secado de punto crítico. A continuación, las muestras se recubren con una fina capa de metal conductor, como el oro, lo que evita el daño térmico de la muestra y proporciona una mejor calidad de imagen.

preparación de muestras para microscopía electrónica

Preparation of Samples for Electron Microscopy

Para ser visualizadas por un microscopio electrónico, ya sea de transmisión o de barrido, las muestras biológicas deben fijarse (estabilizarse) para que el haz de electrones no las destruya y secarse completamente (secar/deshidratar) para que el vacío no las afecte. La fijación debe realizarse lo más rápido posible porque las propiedades de la muestra comenzarán a cambiar tan pronto como se retire de su entorno natural. Por ejemplo, en una muestra de tejido, los niveles de oxígeno comienzan a disminuir, provocando una apariencia alterada de las mitocondrias.

Avances en la preparación de muestras TEM

Criofijación

En esta técnica, las células y orgánulos pequeños se pueden congelar mediante congelación por inmersión, mientras que las células y tejidos más grandes se pueden congelar mediante alta presión. La congelación por inmersión se logra colocando una pequeña cantidad de muestra en una rejilla EM fría y entonces sumergiéndola rápidamente en etano líquido lleno en un depósito y rodeado de nitrógeno líquido. La rápida congelación garantiza que el agua no cristalice. En la congelación a alta presión, se utiliza nitrógeno líquido para congelar la muestra rápidamente y se somete a una presión de ~2000 bar para desacelerar la formación de cristales de hielo. Las muestras criofijadas se seccionan en un crioultramicrotomo que consta de un cuchillo de diamante congelado.

Proyección de sombras

La plataforma giratoria en una cámara de vacío contiene la muestra, que entonces se recubre con metal pesado. La rotación de la muestra garantiza un recubrimiento metálico en la parte frontal y un efecto de sombra en la parte posterior. La técnica es particularmente útil cuando se estudian detalles ultraestructurales de bacterias o virus, ADN, ARN o proteínas aisladas.

Tinción negativa

Se pueden obtener imágenes de muestras pequeñas, como ribosomas, moléculas de enzimas, virus, etc., directamente sin seccionarlas. Las muestras se mezclan con molibdato de amonio, acetato de uranilo o ácido fosfotúngstico, que son opacos a los electrones. Se forma una capa de metal en la rejilla, excepto donde se encuentra la muestra, creando una imagen negativa de la muestra. El contraste se logra como resultado del patrón de distribución del metal.

Preparación de muestras desecadas y no desecadas para SEM

SEM puede obtener imágenes de tres tipos de muestras, a saber, (i) desecadas y conductoras, (ii) desecadas y no conductoras, y (c) no desecadas (húmedas). Las muestras naturalmente conductoras que están desecadas se pueden montar y visualizar fácilmente. Sin embargo, las muestras desecadas y no conductoras deben recubrirse con una capa conductora para proteger la muestra del sobrecalentamiento y mejorar la calidad de la imagen. Estas muestras se vuelven conductoras recubriendo la muestra con una fina capa de oro usando un recubridor por pulverización catódica. Utiliza iones de argón para eliminar los átomos de oro de una placa de oro y cubrir la superficie de la muestra con oro, haciendo que la superficie sea conductora de electrones.

Las muestras húmedas pueden desgasificarse en el ambiente de vacío del microscopio.

El SEM húmedo, también llamado SEM ambiental o ESEM, debe controlar la sublimación del agua en la muestra durante la toma de imágenes. Se logra recubriendo primero la muestra con una capa delgada de un polímero membranoso que es transparente a los haces de electrones y separa la muestra húmeda del vacío. entonces la temperatura disminuye, lo que disminuye la presión de vapor saturado. Estas condiciones permiten la operación del instrumento a un vacío mayor que preserva las características ultraestructurales de la muestra y reduce el ruido. También se puede utilizar un Deben Coolstage para obtener imágenes de muestras húmedas congelándolas a -25 °C, lo que preserva la estructura de la muestra y evita la desgasificación mientras los electrones la bombardean.

Preparación de muestras para microscopía electrónica

Para ser visualizadas por un microscopio electrónico, ya sea de transmisión o de barrido, las muestras biológicas deben fijarse (estabilizarse) para que el haz de electrones ...

preparation-of-samples-for-electron-microscopy

Education

Educación

JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Visualizing Cells, Tissues, and Molecules

Visualización de células, tejidos y moléculas

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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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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 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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Antibody Staining in Cells and Tissues

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Confocal Microscopy for Sharp Cell Images

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Fluorescent Methods for Protein Movement

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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

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Electron Microscopy: TEM vs SEM

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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...

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Immunogold Labeling in Electron Microscopy

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Cryo-EM for Native Biomolecule Imaging

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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 Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

Electron Microscopy Sample Prep Methods

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