Name: fluorescent methods for protein movement
Uploaded: 2023-04-30T12:00:21.000Z
Duration: 1 min 19 s
Channel: JoVE

La dinamica cellulare delle proteine marcate in fluorescenza può essere studiata utilizzando tecniche microscopiche avanzate come FRAP, SPT e FRET.
Nel recupero della fluorescenza dopo il fotosbiancamento o FRAP, una piccola regione contenente proteine di interesse viene irradiata con un raggio laser focalizzato per fotosbiancare i fluorofori.
Quando le proteine marcate si diffondono nell'area sbiancata, la regione diventa di nuovo fluorescente. La velocità di recupero della fluorescenza determina la velocità di diffusione delle proteine.

Il tracciamento di singole particelle o SPT coinvolge proteine marcate con anticorpi legati al fluoroforo. Il movimento delle singole proteine viene monitorato utilizzando un videomicroscopio computerizzato.
Il trasferimento di energia per risonanza di Forster o FRET misura la prossimità di due proteine marcate con fluorofori. Quando la distanza è superiore a dieci nanometri, non si verifica alcuna fluorescenza. Se le proteine si avvicinano, l'eccitazione del fluoroforo donatore porta al trasferimento di energia al fluoroforo accettore.
 Il trasferimento di energia riduce la fluorescenza del dono mentre aumenta l'emissione dell'accettore, permettendone la visualizzazione.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, E.L. e Phair, R.D., 2018. Lo sviluppo e il miglioramento di FRAP come strumento chiave per lo studio della dinamica delle proteine. Rivista biofisica, 115(7), pp.1146-1155.</li>
	<li>Takanishi, CL, Bykova, EA, Cheng, W. e Zheng, J., 2006. Analisi FRET basata su GFP in cellule vive. Ricerca sul cervello, 1091(1), pp.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. e Olcese, R., 2018. Sfruttare il trasferimento di elettroni fotoindotto per determinare otticamente le distanze atomiche su scala sub-nanometrica delle proteine. Comunicazioni sulla natura, 9(1), pp.1-12.</li>
</ol>

dinamica delle proteine nelle cellule viventi

Protein Dynamics in Living Cells

Per studiare la dinamica delle proteine nelle cellule viventi vengono utilizzate diverse tecniche basate sulla fluorescenza. Queste tecniche includono FRAP, FRET e PET.

Il recupero fluorescente dopo il fotosbiancamento (FRAP) è una tecnica di rilevamento basata su proteine fluorescenti utilizzata per quantificare la velocità di movimento delle proteine all'interno della cellula. Questo metodo espone una piccola porzione della cellula a un intenso raggio laser. Il raggio laser provoca il fotosbiancamento permanente delle proteine marcate con fluorofori nella regione esposta. Man mano che le proteine sbiancate si diffondono nel tempo e le proteine marcate con fluorescenza provenienti da altre parti della cellula si spostano verso l'interno, la regione diventa nuovamente fluorescente. La velocità di movimento viene quantificata tracciando l'intensità relativa della fluorescenza rispetto al tempo impiegato, il che consente di determinare la velocità con cui la proteina si muove all'interno della cellula.

Il trasferimento di energia per risonanza Förster (FRET) è una tecnica molecolare utilizzata per determinare la distanza tra due proteine. Pertanto, FRET può essere utilizzato come righello molecolare. La tecnica utilizza due proteine marcate con fluoroforo, una che funge da donatore e l'altra da accettore. Dopo l'eccitazione ad una lunghezza d'onda specifica, il fluoroforo donatore emette energia di fluorescenza che viene assorbita dal fluoroforo accettore. Il fluoroforo donatore ritorna allo stato fondamentale dopo il trasferimento di energia, mentre il fluoroforo accettore emette fluorescenza che viene visualizzata con un microscopio a fluorescenza. La tecnica FRET dipende da tre fattori: la distanza tra le proteine interagenti, l'entità della sovrapposizione spettrale tra i fluorofori donatore e accettore e l'orientamento del fluoroforo donatore e accettore durante il trasferimento di energia. Il trasferimento di energia tra i fluorofori donatori e accettori correttamente orientati può avvenire solo quando la distanza tra le due proteine interagenti è pari o inferiore a 10 nm.

Il trasferimento elettronico fotoindotto o PET determina la distanza subatomica tra le proteine in una cellula. Nel PET, il fluoroforo assorbe la luce ed emette un segnale di fluorescenza attraverso un elettrone eccitato. L'elettrone eccitato viene trasferito al recettore. Durante il trasferimento di energia, avviene una reazione redox, che genera una separazione di carica tra le proteine donatrici e accettrici.

Dinamica delle proteine nelle cellule viventi

Diverse tecniche basate sulla fluorescenza vengono utilizzate per studiare la dinamica delle proteine nelle cellule viventi. Queste tecniche includono FRAP, FRET e PET.
Fluorescente...

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Cell Biology

Biologia cellulare

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Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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 and fluorescent proteins from other parts of the cell move in. The region then becomes fluorescent again, and the change in intensity is plotted against time to calculate how fast the protein moves.

Fӧrster resonance energy transfer, or FRET, measures the distance between two proteins and can act like a molecular ruler. It uses two fluorophore-tagged proteins, a donor and an acceptor. When the donor is excited at a specific wavelength, it transfers fluorescence energy to the acceptor. The donor returns to the ground state, and the acceptor emits fluorescence that can be seen with a fluorescence microscope. FRET depends on distance between the proteins, spectral overlap between the fluorophores, and their orientation during energy transfer. This transfer occurs only when the interacting proteins are 10 nm apart or less.

Photoinduced electron transfer, or PET, measures sub-atomic distance between proteins in a cell. In PET, the fluorophore absorbs light and sends out a fluorescence signal through an excited electron. That electron is transferred to a receptor. During this transfer, a redox reaction creates charge separation between the donor and acceptor proteins.

Fluorescent Methods for Protein Movement

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

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

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 FRAP, FRET, and PET use fluorescence to measure protein movement, protein distance, and protein interactions in living cells.

Fluorescent Methods for Protein Movement | 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.

Fluorescent Methods for Protein Movement

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