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

Amostras biológicas devem ser preparadas para microscopia eletrônica para proteger a amostra de danos causados pelo poderoso feixe de elétrons e pela baixa pressão criada pelo vácuo.
A preparação da amostra começa com a fixação química e desidratação completa. Fixadores químicos, como glutaraldeído ou formaldeído, reticulam as proteínas, enquanto o tetróxido de ósmio é frequentemente usado para fixar os lipídios da membrana.
Em seguida, a amostra é desidratada gradualmente com concentrações crescentes de etanol ou acetona para evitar o encolhimento da amostra.
Para TEM, as amostras são incorporadas em uma resina epóxi dura. Um ultramicrótomo então corta a amostra em seções transversais ultrafinas pelas quais os elétrons podem passar.
Essas seções são manchadas com metais pesados, como acetato de uranila. As moléculas que ligam os metais parecem mais escuras e fornecem contraste à imagem.
Para SEM, as amostras são secas usando dióxido de carbono líquido em um processo chamado secagem de ponto crítico. As amostras são então revestidas com uma fina camada de metal condutor, como ouro, evitando danos térmicos à amostra e proporcionando melhor qualidade de imagem.

preparação de amostras para microscopia eletrônica

Preparation of Samples for Electron Microscopy

Para serem visualizadas por um microscópio eletrônico, seja de transmissão ou de varredura, as amostras biológicas precisam ser fixadas (estabilizadas) para que o feixe de elétrons não as destrua, e bem secas (dessecadas/desidratadas) para que o vácuo não as afete. A fixação precisa ser feita o mais rápido possível, porque as propriedades da amostra começarão a mudar assim que ela for removida do seu ambiente natural. Por exemplo, numa amostra de tecido, os níveis de oxigênio começam a diminuir, causando uma aparência alterada das mitocôndrias. Avanços na preparação de amostras para MET Criofixação Nesta técnica, pequenas células e organelas podem ser congeladas por congelamento por imersão, enquanto células e tecidos maiores podem ser congelados usando alta pressão. O congelamento por imersão é obtido colocando uma pequena quantidade de amostra em uma grade EM fria e, em seguida, mergulhando rapidamente em etano líquido preenchido em um reservatório e cercado por nitrogênio líquido. O congelamento rápido garante que a água não cristalize. No congelamento de alta pressão, o nitrogênio líquido é usado para congelar a amostra rapidamente e é submetido a uma pressão de aproximadamente 2.000 bar para desacelerar a formação de cristais de gelo. As amostras criofixadas são seccionadas em um crio-ultramicrótomo que consiste em uma faca de diamante congelada. Projeção de sombra A plataforma rotativa em uma câmara de vácuo contém a amostra, que é então revestida com metal pesado. A rotação da amostra garante um revestimento metálico na parte frontal e um efeito de sombra na parte traseira. A técnica é particularmente útil no estudo de detalhes ultraestruturais de bactérias ou vírus, DNA, RNA ou proteínas isoladas. Coloração negativa Amostras pequenas, como ribossomos, moléculas de enzimas, vírus, etc., podem ser visualizadas diretamente sem corte. As amostras são misturadas com molibdato de amônio, acetato de uranila ou ácido fosfotúngstico, que são eletronicamente opacos. Uma camada metálica é formada na grade, exceto onde a amostra está, criando uma imagem negativa da amostra. O contraste é obtido como resultado do padrão de distribuição do metal. Preparação de amostras dessecadas e não dessecadas para MEV A MEV pode gerar imagens de três tipos de amostras, a saber, (i) dessecadas e condutoras, (ii) dessecadas e não condutoras e (c) não dessecadas (úmidas). Amostras naturalmente condutoras que são dessecadas podem ser facilmente montadas e fotografadas. No entanto, amostras dessecadas e não condutoras devem ser revestidas com uma camada condutora para proteger a amostra do superaquecimento e melhorar a qualidade da imagem. Essas amostras são tornadas condutoras revestindo a amostra com uma fina camada de ouro usando um revestidor por pulverização catódica. Essa técnica usa íons de argônio para retirar átomos de ouro de uma placa de ouro e revestir a superfície da amostra com ouro, tornando a superfície condutora de elétrons. Amostras úmidas podem liberar gases no ambiente de vácuo do microscópio. A MEV úmida, também chamado MEV Ambiental ou ESEM, deve controlar a sublimação da água na amostra durante a geração de imagens. Isso é conseguido revestindo primeiro a amostra com uma fina camada de um polímero membranoso que é transparente aos feixes de elétrons e separa a amostra úmida do vácuo. Então a temperatura diminui, o que diminui a pressão do vapor saturado. Estas condições permitem a operação do instrumento em um vácuo mais elevado, que preserva as características ultraestruturais da amostra e reduz o ruído. Um Deben Coolstage também pode ser usado para obter imagens de amostras úmidas, congelando-as a -25°C, o que preserva a estrutura da amostra e evita a liberação de gases enquanto os elétrons a bombardeiam.

Preparação de Amostras para Microscopia Eletrônica

Para serem visualizadas por um microscópio eletrônico, seja por transmissão ou varredura, as amostras biológicas precisam ser fixadas (estabilizadas) para que o feixe de elétrons ...

preparation-of-samples-for-electron-microscopy

Education

JoVE Core

Cell Biology

Biologia Celular

Unit 1

Unidade 1

Visualizing Cells, Tissues, and Molecules

Visualizando Células, Tecidos e Moléculas

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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

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

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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...

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: 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

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 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 - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

Electron Microscopy Sample Prep Methods

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