Name: cryo-em for native biomolecule imaging
Uploaded: 2023-04-30T12:01:32.000Z
Duration: 1 min 28 s
Channel: JoVE

クライオ電子顕微鏡またはクライオ電子顕微鏡は、-150°C未満の極低温で凍結した生体サンプルを検査します。
従来のEMとは異なり、クライオEMはサンプルの固定や脱水を必要としないため、生物学的構造の自然な状態を維持するのに役立ちます。
電磁界で使用される高エネルギー電子は、サンプルの化学結合を損傷する可能性があります。サンプルを凍結すると、この損傷を防ぎ、原子解像度に近い画像を得ることができます。
クライオ電子顕微鏡では、溶液中の小さな生体サンプルが液体エタン中でガラス化、つまり急速に凍結されます。ガラス化は、サンプルをその自然な状態で薄い水層に固定し、サンプル中の水は氷の結晶の出現なしにガラスのような状態を形成します。
細胞や組織などの厚いサンプルは、薄い液体層でガラス化することはできません。したがって、これらのサンプルは、ガラス質切片のクライオEM法を使用して研究できます。この方法では、まず高圧凍結により試料をガラス化し、その後-140°Cで極薄切片に切断した後、クライオ電子顕微鏡で観察します。

クライオ電子顕微鏡

Cryo-electron Microscopy

従来の電子顕微鏡 (EM) には、生体サンプルの脱水、固定、および染色が含まれており、これにより生体分子の本来の状態が歪み、いくつかのアーチファクトが生じます。 また、高エネルギーの電子線は試料に損傷を与え、高解像度の画像を取得することが困難になります。 これらの問題は、凍結サンプルとより穏やかな電子ビームを使用するクライオ EM を使用して対処できます。 この技術はジャック・デュボシェ氏、ヨアヒム・フランク氏、リチャード・ヘンダーソン氏によって開発され、2017年にノーベル化学賞を受賞した。

X 線回折および核磁気共鳴 (NMR) 技術は、生体分子の高分解能構造を取得したり、その構造変化を研究したりするためにも使用されます。 ただし、X 線構造を取得するには、分子を結晶化する必要がありますが、毎回結晶化できるわけではありません。 分子が結晶化可能であっても、結晶化プロセスにより生体分子の構造が変化する可能性があり、その生体分子の構造は本来の構造を代表しなくなる可能性があります。 Cryo-EM は結晶化サンプルを必要とせず、天然状態の生体分子の分子運動や相互作用の視覚化も可能にします。 同様に、NMR 技術は比較的小さな可溶性タンパク質のみに限定されており、細胞膜に埋め込まれた非極性タンパク質には適していません。 一方、クライオ EM では、より大きなタンパク質、膜結合受容体、または生体分子複合体の研究が可能になります。

Cryo-EM は、生体分子の構造、機能、相互作用を研究するために生化学で広く使用されています。 ウイルスなどの感染因子は、クライオ EM を使用して特定および研究できます。 たとえば、ブラジルでのジカウイルスの発生時には、潜在的な抗ウイルス薬の標的を特定できるように、クライオ EM を利用してウイルス構造の 3D 画像が作成されました。

従来の電子顕微鏡(EM)は、生体試料の脱水、固定、染色を伴い、生体試料の本来の状態を歪めていました。

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Visualizing Cells, Tissues, and Molecules

細胞・組織・分子の可視化

KEY TERMS AND CONCEPTS

主要用語と概念

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 crystallization works, it can change the biomolecule’s structure so it no longer matches the native form.

Cryo-EM also fills gaps left by nuclear magnetic resonance, or NMR. NMR is mainly used for relatively small soluble proteins and is not suited to non-polar proteins in cell membranes. Cryo-EM can be used to study larger proteins, membrane-bound receptors, and biomolecular complexes.

Researchers use cryo-EM to study biomolecule structure, function, and interactions. It can also help identify and analyze infectious agents such as viruses. During the Zika virus outbreak in Brazil, cryo-EM was used to build a 3D image of the virus so potential antiviral drug targets could be found. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, who shared the 2017 Nobel Prize in Chemistry.

Cryo-EM for Native Biomolecule Imaging

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, 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 cryo-EM images frozen biomolecules in native form, avoids crystal-making limits, and helps study proteins, membranes, and viruses.

Cryo-EM for Native Biomolecule Imaging | JoVE K12

cell biology

JoVE Core 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

Cryo-EM for Native Biomolecule Imaging

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