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

La struttura 3D dei campioni biologici può essere determinata utilizzando due tecniche di microscopia crioelettronica: la tomografia elettronica e la ricostruzione di singole particelle.
Nella tomografia elettronica, un campione viene progressivamente inclinato rispetto al fascio di elettroni, catturando una serie di immagini 2D da diverse angolazioni. Queste immagini 2D vengono quindi impilate utilizzando un software per computer per generare l'immagine 3D o il tomogramma del campione.
La tomografia elettronica mantiene l'integrità strutturale in vivo del campione e viene utilizzata principalmente per la visualizzazione 3D di cellule, organelli e macromolecole nel loro ambiente nativo.
Nella ricostruzione di una singola particella, i campioni isolati in vitro, come un campione di proteine purificate, sono distribuiti uniformemente su una griglia.
Viene catturata un'immagine 2D delle singole molecole proteiche che giacciono con orientamenti casuali. Migliaia di tali immagini vengono quindi mediate e allineate per ricostruire l'immagine 3D del campione.

tomografia al microscopio elettronico e ricostruzione di singole particelle

Electron Microscope Tomography and Single-particle Reconstruction

La microscopia elettronica a trasmissione (TEM) può essere utilizzata per determinare la struttura 3D di campioni biologici con l'aiuto di tecniche come la tomografia al microscopio elettronico e la ricostruzione di singole particelle. Mentre la ricostruzione di una singola particella può esaminare macromolecole e complessi macromolecolari solo in condizioni in vitro, la tomografia consente lo studio di componenti cellulari o piccole cellule in vivo.

Tomografia elettronica

La tomografia elettronica può essere eseguita in modalità TEM o STEM (microscopia elettronica a scansione e trasmissione). STEM è una tecnica utilizzata principalmente per ottenere tomogrammi di campioni biologici spessi. Combina la metodologia di scansione della superficie del campione della microscopia elettronica a scansione, in modo tale che gli elettroni trasmessi o dispersi in ciascun punto in cui il fascio di elettroni colpisce il campione vengono raccolti da una serie di rilevatori.

Una variante della tecnica di tomografia elettronica convenzionale è la tomografia a doppio asse, in cui il campione è inclinato attorno a due assi rispetto al fascio di elettroni. Ciò si traduce in due tomogrammi separati che vengono poi allineati per generare un'immagine 3D del campione. La tomografia a doppio asse presenta due vantaggi rispetto alla tomografia a singolo asse: una migliore ricostruzione delle caratteristiche espanse del campione e una maggiore risoluzione della profondità del campione.

Un'altra variante è la criotomografia elettronica, in cui i campioni vengono sottoposti a imaging in condizioni criogeniche perché la fissazione e la disidratazione possono danneggiare le strutture biologiche. Il metodo viene utilizzato principalmente per campioni sottili, di spessore inferiore a 500 nm, poiché i campioni spessi bloccano il fascio di elettroni. Pertanto, la tecnica è stata limitata a complessi macromolecolari purificati, virus o piccole cellule come le cellule batteriche.

Ricostruzione di una singola particella

L'analisi di singole particelle viene generalmente utilizzata con la microscopia crioelettronica per generare strutture 3D con una risoluzione quasi atomica. È più adatto per complessi macromolecolari grandi o dinamici difficili da cristallizzare, rendendolo un sostituto della cristallografia a raggi X. Per i complessi che possono essere cristallizzati, l'analisi di singole particelle è un metodo di scelta per ottenere strutture ad alta risoluzione. La tecnica è stata utilizzata per studiare proteine di membrana, complessi proteici, struttura della cromatina e macchine macromolecolari come ribosomi e proteasomi.

Tomografia al microscopio elettronico e ricostruzione di singole particelle

La microscopia elettronica a trasmissione (TEM) può essere utilizzata per determinare la struttura 3D di campioni biologici con l'aiuto di tecniche come ...

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

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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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