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

Total internal reflection fluorescence microscopy, or TIRF, selectively images fluorescent molecules in a sample close to a solid surface with a higher refractive index, such as a glass coverslip.
The excitation laser beam hits the coverslip at a critical angle, resulting in total internal reflection of the beam.
The reflected light does not enter the sample but creates a narrow electromagnetic field or an evanescent wave, which only illuminates fluorophores within 100 to 200 nanometers of the cell surface.
Because the evanescent wave rapidly decays as it moves further into the sample, most background fluorophores are not excited. So, the resulting images of the few fluorophore-tagged proteins near the solid surface are clear and sharp.
TIRF is routinely used for live-cell imaging, such as studying the movement of motor proteins like kinesin over the microtubules attached to the coverslip.

totale interne reflectie fluorescentiemicroscopie

Total Internal Reflection Fluorescence Microscopy

Totale interne reflectie fluorescentiemicroscopie of TIRF is een geavanceerde microscopische techniek die wordt gebruikt om fluoroforen te visualiseren in monsters dichtbij een vast oppervlak met een hogere brekingsindex, zoals een glazen dekglaasje. TIRF maakt het alleen mogelijk dat fluoroforen in de nabijheid van het vaste oppervlak worden geëxciteerd. Wanneer licht van een medium met een lagere brekingsindex (zoals lucht) onder een kritische hoek het glazen dekglaasje raakt, ondergaat het licht totale interne reflectie in plaats van door het glas te gaan. Dit gebeurt omdat het monster een lagere brekingsindex heeft dan het dekglaasje en het licht niet binnenlaat. Het licht wordt gereflecteerd door het grensvlak en vormt een elektromagnetisch veld dat korte, verdwijnende golven uitzendt. Deze golven prikkelen de fluorofoor alleen dichtbij het oppervlak, omdat ze zich slechts ongeveer 100 tot 200 nm diep in de cel kunnen verplaatsen voordat ze uitsterven.

Er zijn twee soorten TIRF; prisma-gebaseerd en objectief-gebaseerd. Bij op prisma's gebaseerde TIRF-microscopie wordt een prisma op het dekglaasje geplaatst dat de verdwijnende golf naar het monster richt. Bij op objectieven gebaseerde TIRF-microscopie is er geen prisma op de interfase; het doel is hetzelfde als de lichtbron die helpt bij het creëren van de verdwijnende golf.

TIRF heeft verschillende voordelen ten opzichte van traditionele fluorescentie- en confocale microscopen; het voorkomt de verlichting van achtergrondfluoroforen. Het helpt bij het bestuderen van de structuren dicht bij het celoppervlak. Het vermindert het vervagingseffect en zorgt ervoor dat onscherp licht het beeld niet verstoort. Omdat de monsters niet direct worden blootgesteld aan een intense lichtstraal, is de fotobleking minimaal en worden de cellen minder blootgesteld aan fototoxiciteit.

Totale interne reflectie fluorescentiemicroscopie

Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid ...

total-internal-reflection-fluorescence-microscopy

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Cell Biology

Celbiologie

Unit 1

Eenheid 1

Visualizing Cells, Tissues, and Molecules

Visualisatie van cellen, weefsels en moleculen

KEY TERMS AND CONCEPTS

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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

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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

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Surface-Only Imaging with TIRF Microscopy

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