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

生物样本必须经过制备才能用于电子显微镜观察，以保护样本免受强电子束以及真空所产生低压的损伤。
样品制备始于化学固定和彻底脱水。化学固定剂（如戊二醛或甲醛）可交联蛋白质，而四氧化锇常用于固定膜脂质。
接下来，使用浓度逐渐增加的乙醇或丙酮对样品进行逐步脱水，以避免样品收缩。
进行透射电镜观察时，样品被包埋在硬质环氧树脂中。随后，超薄切片机将样品切成超薄的横截面，以便电子能够穿透。
这些切片使用醋酸铀等重金属进行染色。结合金属的分子呈现较深的色泽，从而为图像提供对比度。
对于扫描电镜观察，样品需通过临界点干燥法，使用液态二氧化碳进一步干燥。随后，样品表面溅射镀上一层薄的导电金属（如金），以防止样品受热损伤，并提高成像质量。

电子显微镜样品的制备

Preparation of Samples for Electron Microscopy

为了在透射电子显微镜或扫描电子显微镜下观察，生物样品需要被固定（稳定）以防止电子束破坏它们，并彻底干燥（干燥/脱水）以防止真空对它们产生影响。固定需要尽快完成，因为一旦样品从自然环境中移出，其特性就会开始发生变化。 例如，在组织样本中，氧含量开始下降，导致线粒体外观发生改变。

TEM 样品制备的进展

冷冻固定

在这项技术中，小的细胞和细胞器可以通过快速冷冻法进行冷冻，而较大的细胞和组织可以使用高压来冷冻。快速冷冻是通过将一小部分样品放在冷的电磁网格上，然后迅速浸入充满液态乙烷的储液器（被液氮包围）中实现的。快速冷冻可确保水不会结晶。 在高压冷冻中，液氮用于快速冷冻样品，并承受约 2000 bar 的压力以减缓冰晶的形成。冷冻固定的样本在冷冻超薄切片机中用冷冻钻石刀进行切片。

阴影投射

真空室中的旋转平台固定着样品，然后在样品上涂上一层重金属。样品的旋转确保了正面有一层金属涂层，背面产生阴影效果。该技术在研究细菌或病毒、DNA、RNA 或分离蛋白质的超微结构细节时特别有用。

负染色

小样品，如核糖体、酶分子、病毒等，无需切片即可直接成像。样品与钼酸铵、醋酸铀或磷钨酸等电子不透明物质混合。除了样品所在的位置之外，金属层会形成在网格上，从而形成样品的负像。对比度是通过金属分布模式实现的。

为SEM准备干燥和未干燥样本

SEM 可以对三种类型的样品进行成像，即 (i) 干燥且导电，(ii) 干燥且不导电，以及 (c) 非干燥（湿）。自然导电的干燥样品可以很容易地安装和成像。然而，干燥且不导电的样品必须涂有导电层，以防止样品过热并提高图像质量。通过使用溅射镀膜机在样品上涂上一层薄金层，使这些样品具有导电性。溅射镀膜机使用氩离子从金板上击落金原子并覆盖样品表面，使表面导电。

湿样品会在显微镜的真空环境中释放气体。

湿式 SEM，也称为环境 SEM 或 ESEM，必须在成像过程中控制样品中的水分升华。这首先通过在样本上涂上一层对电子束透明的膜状聚合物来实现，这层聚合物将湿样本与真空隔离开来。然后降低温度，降低饱和蒸气压。这些条件允许仪器在更高的真空下运行，从而保留样本的超微结构特征并降低噪音。Deben Coolstage 还可用于通过将湿样品冷冻至 -25°C 来对其进行成像，这样可以保留样品结构并防止电子轰击时释放气体。

要通过透射或扫描电子显微镜进行观察，生物样品需经过固定（稳定化）处理，以便电子束不会...

preparation-of-samples-for-electron-microscopy

Education

教育

JoVE Core

Cell Biology

细胞生物学

Unit 1

第一单元

Visualizing Cells, Tissues, and Molecules

细胞、组织与分子的可视化

KEY TERMS AND CONCEPTS

关键术语与概念

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

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

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 Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Electron Microscopy Sample Prep Methods

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