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

يصور الفحص المجهري الفلوري للانعكاس الداخلي الكلي ، أو TIRF ، بشكل انتقائي جزيئات الفلورسنت في عينة قريبة من سطح صلب مع معامل انكسار أعلى ، مثل غطاء زجاجي.
يضرب شعاع ليزر الإثارة الغطاء بزاوية حرجة ، مما يؤدي إلى انعكاس داخلي كامل للحزمة.
لا يدخل الضوء المنعكس إلى العينة ولكنه يخلق مجالا كهرومغناطيسيا ضيقا أو موجة زائلة ، والتي تضيء فقط الفلوروفورات في نطاق 100 إلى 200 نانومتر من سطح الخلية.
نظرا لأن الموجة الزائلة تتحلل بسرعة كلما تحركت أكثر في العينة ، فإن معظم الفلوروفورات في الخلفية ليست متحمسة. لذا ، فإن الصور الناتجة عن عدد قليل من البروتينات الموسومة بالفلوروفور بالقرب من السطح الصلب واضحة وحادة.
يستخدم TIRF بشكل روتيني لتصوير الخلايا الحية ، مثل دراسة حركة البروتينات الحركية مثل كينيسين فوق الأنابيب الدقيقة المتصلة بالغطاء.

<ol>
	<li>فيش ، ك. ن. ، 2009. الفحص المجهري الكامل للانعكاس الداخلي (TIRF). البروتوكولات الحالية في القياس الخلوي ، 50 (1) ، ص 12-18.</li>
</ol>

المجهر الفلوري الانعكاسي الداخلي الكلي

Total Internal Reflection Fluorescence Microscopy

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المجهر الفلوري الانعكاسي الداخلي الكلي أو TIRF هو تقنية مجهرية متقدمة تستخدم لتصور الفلوروفورات في عينات قريبة من سطح صلب مع معامل انكسار أعلى، مثل ساترة زجاجية. يسمح TIRF فقط بإثارة الفلوروفورات القريبة من السطح الصلب. عندما يضرب الضوء من وسط ذو معامل انكسار أقل (مثل الهواء) الغطاء الزجاجي بزاوية حرجة، فإن الضوء يخضع لانعكاس داخلي كلي بدلاً من المرور عبر الزجاج. يحدث هذا لأن العينة تحتوي على معامل انكسار أقل من الغطاء ولا تسمح للضوء بالدخول إلى الداخل. ينعكس الضوء من الواجهة ويشكل مجالًا كهرومغناطيسيًا ينبعث منه موجات متلاشية قصيرة الطول. تثير هذه الموجات فقط الفلوروفور بالقرب من السطح حيث يمكنها التحرك لمسافة تتراوح بين 100 إلى 200 نانومتر فقط داخل الخلية قبل أن تموت.

هناك نوعان من TIRF؛ على أساس المنشور وعلى أساس الهدف. في الفحص المجهري TIRF القائم على المنشور، يتم وضع المنشور على سطح ساترة الذي يوجه الموجة الزائلة إلى العينة. في الفحص المجهري TIRF القائم على الهدف، لا يوجد منشور في الطور البيني؛ الهدف هو نفس مصدر الضوء الذي يساعد في إنشاء الموجة الزائلة.

يتمتع TIRF بالعديد من المزايا مقارنة بالمجاهر التقليدية والمجاهر البؤرية؛ يمنع إضاءة الفلوروفورات الخلفية. يساعد في دراسة الهياكل القريبة من سطح الخلية. فهو يقلل من تأثير التمويه ولا يسمح للضوء خارج نطاق التركيز بالتداخل مع الصورة. وبما أن العينات لا تتعرض مباشرة لشعاع ضوء مكثف، فإن التبييض الضوئي هو الحد الأدنى، وتكون الخلايا أقل تعرضًا للسمية الضوئية.

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الفحص المجهري الفلوري للانعكاس الداخلي الكلي أو TIRF هو تقنية مجهرية متقدمة تستخدم لتصور الفلوروفورات في عينات قريبة من العينات الصلبة ...

total-internal-reflection-fluorescence-microscopy

Education

JoVE Core

Cell Biology

بيولوجيا الخلية

Unit 1

الوحدة الأولى

Visualizing Cells, Tissues, and Molecules

تصوير الخلايا والأنسجة والجزيئات

KEY TERMS AND CONCEPTS

المصطلحات والمفاهيم الأساسية

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

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...

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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

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...

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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, 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...

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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

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transmission electron microscopy

How TEM Reveals Tiny Structures

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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...

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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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