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

생물학적 샘플은 강력한 전자빔과 진공에 의해 생성된 낮은 압력으로 인한 손상으로부터 샘플을 보호하기 위해 전자 현미경을 위해 준비되어야 합니다.
시료 준비는 화학적 고정과 완전한 탈수로 시작됩니다. 글루타르알데히드 또는 포름알데히드와 같은 화학적 고정제는 단백질을 가교하는 반면, 사산화오스뮴은 막 지질을 고정하는 데 자주 사용됩니다.
다음으로, 샘플 수축을 방지하기 위해 에탄올 또는 아세톤의 농도가 증가함에 따라 샘플을 점진적으로 탈수합니다.
TEM의 경우 샘플이 경질 에폭시 수지에 내장되어 있습니다. 그런 다음 초박 절개기는 샘플을 전자가 통과할 수 있는 초박형 단면으로 자릅니다.
이 섹션은 우라닐 아세테이트와 같은 중금속으로 염색되어 있습니다. 금속을 결합하는 분자는 더 어둡게 나타나고 이미지에 대비를 제공합니다.
SEM의 경우, 임계점 건조라는 공정에서 액체 이산화탄소를 사용하여 샘플을 추가로 건조합니다. 그런 다음 샘플에 금과 같은 전도성 금속의 얇은 층을 스퍼터 코팅하여 샘플의 열 손상을 방지하고 더 나은 이미지 품질을 제공합니다.

전자현미경용 표본 준비

Preparation of Samples for Electron Microscopy

전자현미경(투과 또는 스캐닝)으로 시각화하려면 생물학적 시료를 전자빔이 파괴하지 않도록 고정(안정화)하고 진공이 영향을 주지 않도록 완전히 건조(건조/탈수)해야 합니다. 표본의 특성은 자연 환경에서 제거되자마자 변경되기 시작하므로 가능한 한 빨리 고정을 완료해야 합니다. 예를 들어, 조직 표본에서 산소 수준이 감소하기 시작하여 미토콘드리아의 모양이 변경됩니다.

TEM 표본 준비의 발전

냉동고정

이 기술에서는 작은 세포와 세포소기관은 플런지 냉동을 통해 냉동할 수 있고, 큰 세포와 조직은 고압을 사용하여 냉동할 수 있습니다. 급락 동결은 차가운 EM 그리드에 소량의 표본을 놓은 다음 저장소에 채워지고 액체 질소로 둘러싸인 액체 에탄에 빠르게 급락함으로써 달성됩니다. 급속 냉동으로 인해 물이 결정화되지 않습니다. 고압 냉동에서는 액체 질소를 사용하여 표본을 빠르게 냉동하고 얼음 결정 형성을 늦추기 위해 ~2000bar의 압력을 가합니다. 극저온 고정된 표본은 냉동 다이아몬드 칼로 구성된 극저온 초극절절기로 절단됩니다.

그림자 투사

진공 챔버의 회전 플랫폼은 표본을 보유하고 있으며, 표본은 중금속으로 코팅됩니다. 표본의 회전으로 인해 앞면에는 금속 코팅이, 뒷면에는 그림자 효과가 나타납니다. 이 기술은 박테리아나 바이러스, DNA, RNA 또는 분리된 단백질의 미세 구조 세부 사항을 연구할 때 특히 유용합니다.

네거티브 염색

리보솜, 효소 분자, 바이러스 등과 같은 작은 표본을 분할하지 않고 직접 이미지화할 수 있습니다. 표본은 전자 불투명한 몰리브덴산 암모늄, 우라닐 아세테이트 또는 인텅스텐산과 혼합됩니다. 표본이 있는 곳을 제외하고 그리드에 금속층이 형성되어 표본의 네거티브 이미지가 생성됩니다. 대비는 금속 분포 패턴의 결과로 달성됩니다.

SEM을 위한 건조된 표본과 건조되지 않은 표본 준비

SEM은 세 가지 유형의 표본, 즉 (i) 건조 및 전도성, (ii) 건조 및 비전도성, (c) 비건조(습식) 표본을 이미지화할 수 있습니다. 건조된 자연 전도성 표본을 쉽게 장착하고 이미지화할 수 있습니다. 그러나 건조된 비전도성 표본은 표본이 과열되는 것을 방지하고 이미지 품질을 향상시키기 위해 전도성 층으로 코팅해야 합니다. 이러한 표본은 스퍼터 코터를 사용하여 얇은 금 층으로 표본을 코팅하여 전도성을 갖습니다. 아르곤 이온을 사용하여 금판에서 금 원자를 떨어뜨리고 표본 표면을 금으로 코팅하여 표면을 전자 전도성으로 만듭니다.

젖은 표본은 현미경의 진공 환경에서 가스를 방출할 수 있습니다.

환경 SEM 또는 ESEM이라고도 불리는 습식 SEM은 이미징 중에 표본의 물 승화를 제어해야 합니다. 이는 먼저 전자빔에 투명하고 젖은 표본을 진공에서 분리하는 막형 폴리머의 얇은 층으로 표본을 코팅함으로써 수행됩니다. 그런 다음 온도가 감소하여 포화 증기압이 감소합니다. 이러한 조건을 통해 시편의 미세구조적 특징을 보존하고 소음을 줄이는 더 높은 진공에서 기기를 작동할 수 있습니다. Deben Coolstage는 젖은 표본을 -25°C로 동결하여 이미지화하는 데에도 사용할 수 있습니다. 이는 표본의 구조를 보존하고 전자가 충격을 가하는 동안 가스 방출을 방지합니다.

전자 현미경으로 시각화하려면 투과 또는 스캐닝에서 생물학적 샘플을 고정(안정화)하여 전자빔이 ...

preparation-of-samples-for-electron-microscopy

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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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