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

Toplam iç yansıma floresan mikroskobu veya TIRF, cam lamel gibi daha yüksek bir kırılma indisine sahip katı bir yüzeye yakın bir numunedeki floresan moleküllerini seçici olarak görüntüler.
Uyarma lazer ışını, lamel üzerine kritik bir açıyla çarpar ve ışının toplam iç yansımasına neden olur.
Yansıyan ışık numuneye girmez, ancak hücre yüzeyinin yalnızca 100 ila 200 nanometre içindeki floroforları aydınlatan dar bir elektromanyetik alan veya buharlaşan bir dalga oluşturur.
Buharlaşma dalgası, numunenin içine doğru ilerledikçe hızla bozulduğundan, çoğu arka plan floroforu uyarılmaz. Bu nedenle, katı yüzeye yakın birkaç florofor etiketli proteinin ortaya çıkan görüntüleri net ve keskindir.
TIRF, kinesin gibi motor proteinlerin lamel üzerine bağlı mikrotübüller üzerindeki hareketini incelemek gibi canlı hücre görüntüleme için rutin olarak kullanılır.

<ol>
	<li>Balık, K.N., 2009. Toplam iç yansıma floresan (TIRF) mikroskobu. Sitometride güncel protokoller, 50(1), s.12-18.</li>
</ol>

tam İç yansımalı floresan mikroskobu

Total Internal Reflection Fluorescence Microscopy

Tam iç yansımalı floresan mikroskobu veya TIRF, cam lamel gibi daha yüksek kırılma indeksine sahip katı bir yüzeye yakın numunelerdeki floroforları görselleştirmek için kullanılan gelişmiş bir mikroskopik tekniktir. TIRF yalnızca katı yüzeye yakın olan floroforların uyarılmasına izin verir. Kırılma indisi daha düşük olan bir ortamdan (hava gibi) gelen ışık, cam lamel üzerine kritik bir açıyla çarptığında, ışık camdan geçmek yerine tam iç yansımaya uğrar. Bu, numunenin lamelden daha düşük bir kırılma indisine sahip olması ve ışığın içeriye girmesine izin vermemesi nedeniyle gerçekleşir. Işık arayüzden yansır ve kısa uzunlukta uçucu dalgalar yayan bir elektromanyetik alan oluşturur. Bu dalgalar, ölmeden önce hücre içinde yalnızca yaklaşık 100 ila 200 nm derinlikte hareket edebildikleri için yalnızca yüzeye yakın floroforu uyarır. İki tür TIRF vardır; prizma temelli ve nesnel temelli. Prizma bazlı TIRF mikroskobunda lamel yüzeyine, fani dalgayı numuneye yönlendiren bir prizma yerleştirilir. Objektif tabanlı TIRF mikroskobunda interfazda prizma yoktur; amaç, kaybolan dalgayı yaratmaya yardımcı olan ışık kaynağıyla aynıdır. TIRF'in geleneksel floresans ve eş odaklı mikroskoplara göre birçok avantajı vardır; arka plandaki floroforların aydınlatılmasını engeller. Hücre yüzeyine yakın yapıların incelenmesine yardımcı olur. Bulanıklaştırma etkisini azaltır ve odak dışı ışığın görüntüye müdahale etmesine izin vermez. Numuneler doğrudan yoğun bir ışık ışınına maruz kalmadığından, ışıkla ağartma minimum düzeydedir ve hücreler fototoksisiteye daha az maruz kalır.

Tam İç Yansımalı Floresan Mikroskobu

Toplam iç yansıma floresan mikroskobu veya TIRF, katı bir yapıya yakın numunelerdeki floroforları görselleştirmek için kullanılan gelişmiş bir mikroskobik tekniktir.

total-internal-reflection-fluorescence-microscopy

Education

JoVE Core

Cell Biology

Hücre Biyolojisi

Unit 1

Ünite 1

Visualizing Cells, Tissues, and Molecules

Hücrelerin, Dokuların ve Moleküllerin Görülebilir Kılınması

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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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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...

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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 Hücre Biyolojisi, anlaşılması kolay ancak kapsamlı animasyonlu video derslerde karmaşık hücresel süreçleri gösterir. Bu koleksiyon, organeller ve zarlar, hücresel taşıma ve hücre enerjileri dahil olmak üzere hücrelerin temel bileşenlerine ve işlevlerine odaklanır. Gerçek dünya araştırma deneylerinde hücre biyolojisinin uygulanmasını gösteren bilim insanı eylem videoları ile tamamlanmaktadır.

Surface-Only Imaging with TIRF Microscopy

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