Name: 3d imaging with electron microscopy
Uploaded: 2023-04-30T12:01:40.000Z
Duration: 1 min 7 s
Channel: JoVE

생물학적 샘플의 3D 구조는 전자 단층 촬영과 단일 입자 재구성이라는 두 가지 초저온 전자 현미경 기술을 사용하여 결정할 수 있습니다.
전자 단층 촬영에서 샘플은 전자빔에 비해 점진적으로 기울어져 다양한 각도에서 일련의 2D 이미지를 캡처합니다. 그런 다음 컴퓨터 소프트웨어를 사용하여 이러한 2D 이미지를 쌓아 샘플의 3D 이미지 또는 단층 사진을 생성합니다.
전자 단층촬영은 샘플의 생체 내 구조적 무결성을 유지하며 주로 기본 환경에서 세포, 소기관 및 거대분자의 3D 시각화에 사용됩니다.
단일 입자 재구성에서는 정제된 단백질 샘플과 같은 분리된 체외 샘플이 그리드에 고르게 분포되어 있습니다.
임의의 방향으로 놓여 있는 개별 단백질 분자의 2D 이미지가 캡처됩니다. 그런 다음 수천 개의 이미지를 평균화하고 정렬하여 샘플의 3D 이미지를 재구성합니다.

전자현미경 단층촬영 및 단일 입자 재구성

Electron Microscope Tomography and Single-particle Reconstruction

투과전자현미경(TEM)은 전자현미경 단층촬영 및 단일 입자 재구성과 같은 기술의 도움으로 생물학적 시료의 3D 구조를 결정하는 데 사용할 수 있습니다. 단일 입자 재구성은 시험관 내 조건에서만 거대분자와 거대분자 복합체를 검사할 수 있는 반면, 단층 촬영은 생체 내에서 세포 구성 요소 또는 작은 세포에 대한 연구를 허용합니다.

전자단층촬영

전자단층촬영은 TEM 또는 STEM(주사 투과 전자 현미경) 모드에서 수행할 수 있습니다. STEM은 두꺼운 생물학적 표본의 단층촬영을 얻는 데 주로 사용되는 기술입니다. 이는 주사 전자 현미경의 시료 표면 스캐닝 방법을 결합하여 전자 빔이 시료에 닿는 각 지점에서 전송되거나 산란된 전자가 일련의 검출기에 의해 수집됩니다.

기존 전자 단층 촬영 기술의 변형은 이중 축 단층 촬영으로, 여기서 표본은 전자 빔에 대해 두 축을 중심으로 기울어집니다. 이로 인해 표본의 3D 이미지를 생성하기 위해 정렬된 두 개의 개별 단층촬영이 생성됩니다. 이중 축 단층 촬영에는 단일 축 단층 촬영에 비해 두 가지 장점이 있습니다. 즉, 표본의 확장된 특징을 더 잘 재구성하고 표본 깊이 분해능이 향상된다는 것입니다.

또 다른 변형은 고정 및 탈수가 생물학적 구조를 손상시킬 수 있기 때문에 극저온 조건에서 표본을 이미지화하는 전자 저온 단층 촬영입니다. 이 방법은 두꺼운 시료가 전자빔을 차단하기 때문에 두께가 500 nm 미만인 얇은 시료에 주로 사용됩니다. 따라서 이 기술은 정제된 거대분자 복합체, 바이러스 또는 박테리아 세포와 같은 작은 세포에만 국한되어 왔습니다.

단일 입자 재구성

단일 입자 분석은 일반적으로 극저온 전자 현미경과 함께 사용되어 원자에 가까운 분해능으로 3D 구조를 생성합니다. 이는 결정화가 어려운 크고 동적 거대분자 복합체에 가장 적합하므로 X선 결정학을 대체할 수 있습니다. 결정화될 수 있는 복합체의 경우 단일 입자 분석은 고해상도 구조를 얻기 위해 선택하는 방법입니다. 이 기술은 막 단백질, 단백질 복합체, 염색질 구조, 리보솜 및 프로테아솜과 같은 거대분자 기계를 연구하는 데 사용되었습니다.

투과 전자 현미경 (TEM)은 전자와 같은 기술의 도움으로 생물학적 샘플의 3D 구조를 결정하는 데 사용할 수 있습니다.

electron-microscope-tomography-and-single-particle-reconstruction

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

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 combines sample scanning with detectors that collect electrons transmitted or scattered where the beam hits the sample.

A common variation is dual-axis tomography. In this method, the sample is tilted around two axes relative to the electron beam. The two tomograms are then aligned to form a 3D image. Dual-axis tomography can improve the reconstruction of expanded features and increase depth resolution compared with single-axis tomography.

Another form is electron cryo-tomography. Samples are imaged under cryogenic conditions because fixation and dehydration can damage biological structures. This method is mainly used for thin samples, usually less than 500 nm thick, because thicker samples block the electron beam. It has therefore been limited to purified macromolecular complexes, viruses, and small cells such as bacterial cells.

Single-particle analysis is usually paired with cryo-electron microscopy to produce 3D structures with near-atomic resolution. It works best for large or dynamic macromolecular complexes that are hard to crystallize, so it can serve as an alternative to X-ray crystallography. When complexes can be crystallized, single-particle analysis is still a method of choice for obtaining high-resolution structures. It has been used to study membrane proteins, protein complexes, chromatin structure, and macromolecular machines such as ribosomes and proteasomes.

3D Imaging with Electron Microscopy

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

preparation-of-samples-for-electron-microscopy

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...

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

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 TEM creates 3D biological images using tomography and single-particle reconstruction, including STEM, dual-axis, and cryo-tomography methods.

3D Imaging with Electron Microscopy | 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.

3D Imaging with Electron Microscopy

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