Name: surface-only imaging with tirf microscopy
Uploaded: 2023-04-30T12:00:29.000Z
Duration: 1 min 5 s
Channel: JoVE

Mikroskopia fluorescencyjna z pełnym wewnętrznym odbiciem (TIRF) selektywnie obrazuje cząsteczki fluorescencyjne w próbce znajdującej się blisko powierzchni ciała stałego o wyższym współczynniku załamania światła, takiej jak szklane szkiełko nakrywkowe.
Wiązka lasera wzbudzającego uderza w szkiełko nakrywkowe pod krytycznym kątem, co powoduje całkowite wewnętrzne odbicie wiązki.
Odbite światło nie dostaje się do próbki, ale tworzy wąskie pole elektromagnetyczne lub falę ulotną, która oświetla fluorofory tylko w promieniu od 100 do 200 nanometrów od powierzchni komórki.
Ponieważ fala ulotna szybko zanika w miarę przemieszczania się w głąb próbki, większość fluoroforów tła nie jest wzbudzona. Tak więc uzyskane obrazy kilku białek znakowanych fluoroforem w pobliżu powierzchni ciała stałego są wyraźne i ostre.
TIRF jest rutynowo stosowany do obrazowania żywych komórek, takich jak badanie ruchu białek motorycznych, takich jak kinezyna, nad mikrotubulami przymocowanymi do szkiełka nakrywkowego.

<ol>
	<li>Ryba, K.N., 2009. Mikroskopia fluorescencji całkowitego wewnętrznego odbicia (TIRF). Aktualne protokoły w cytometrii, 50(1), s.12-18.</li>
</ol>

mikroskopia fluorescencyjna z całkowitym odbiciem wewnętrznym

Total Internal Reflection Fluorescence Microscopy

Mikroskopia fluorescencyjna z całkowitym odbiciem wewnętrznym, w skrócie TIRF, to zaawansowana technika mikroskopowa stosowana do wizualizacji fluoroforów w próbkach znajdujących się blisko powierzchni stałej o wyższym współczynniku załamania światła, takiej jak szklane szkiełko nakrywkowe. TIRF pozwala na wzbudzenie jedynie fluoroforów znajdujących się w pobliżu powierzchni stałej. Kiedy światło z ośrodka o niższym współczynniku załamania światła (takiego jak powietrze) uderza w szklane szkiełko nakrywkowe pod krytycznym kątem, światło ulega całkowitemu wewnętrznemu odbiciu, a nie przechodzi przez szkło. Dzieje się tak, ponieważ próbka ma niższy współczynnik załamania światła niż szkiełko nakrywkowe i nie przepuszcza światła. Światło odbija się od granicy faz i tworzy pole elektromagnetyczne emitujące krótkotrwałe fale zanikające. Fale te wzbudzają fluorofor tylko w pobliżu powierzchni, ponieważ mogą przemieszczać się w głąb komórki jedynie na głębokość około 100 do 200 nm, zanim zanikną. Istnieją dwa typy TIRF; oparte na pryzmacie i obiektywne. W mikroskopii pryzmatycznej TIRF na powierzchni szkiełka nakrywkowego umieszcza się pryzmat, który kieruje zanikającą falę w stronę próbki. W obiektywnej mikroskopii TIRF w interfazie nie ma pryzmatu; cel jest taki sam, jak źródło światła, które pomaga wytworzyć zanikającą falę. TIRF ma kilka zalet w porównaniu z tradycyjnymi mikroskopami fluorescencyjnymi i konfokalnymi; zapobiega świeceniu fluoroforów tła. Pomaga w badaniu struktur znajdujących się blisko powierzchni komórki. Redukuje efekt rozmycia i nie pozwala, aby nieostre światło zakłócało obraz. Ponieważ próbki nie są bezpośrednio wystawione na działanie intensywnej wiązki światła, fotowybielanie jest minimalne, a komórki są mniej narażone na fototoksyczność.

Mikroskopia fluorescencyjna z całkowitym odbiciem wewnętrznym

Mikroskopia fluorescencyjna z całkowitym wewnętrznym odbiciem lub TIRF to zaawansowana technika mikroskopowa stosowana do wizualizacji fluoroforów w próbkach zbliżonych do ciała stałego ...

total-internal-reflection-fluorescence-microscopy

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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 electromagnetic field that gives off short evanescent waves. These waves excite only fluorophores near the surface and usually reach about 100 to 200 nm into the cell before fading away.

There are two types of TIRF microscopy: prism-based and objective-based. In prism-based TIRF, a prism sits on the coverslip surface and directs the evanescent wave to the sample. In objective-based TIRF, there is no prism at the interface. The objective itself acts as the light source and helps create the evanescent wave.

TIRF has several advantages over traditional fluorescence and confocal microscopes. It limits the illumination of background fluorophores and helps reduce blur from out-of-focus light. It is especially useful for studying structures close to the cell surface. Because the sample is not exposed to an intense light beam directly, photobleaching is low and the cells experience less phototoxicity.

Surface-Only Imaging with TIRF Microscopy

surface-only-imaging-with-tirf-microscopy

imaging biological samples with optical microscopy

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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...

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Microscope Contrast for Live, Unstained Cells

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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...

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Fluorescent Antibody Staining in Cells

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immunocytochemistry and immunohistochemistry

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Antibody Staining in Cells and Tissues

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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...

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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, 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...

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scanning electron microscopy

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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

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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...

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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

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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 TIRF microscopy uses evanescent waves to excite fluorophores near a glass surface, with prism-based and objective-based setups.

Surface-Only Imaging with TIRF 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.

Surface-Only Imaging with TIRF Microscopy

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