Total Internal Reflection Fluorescence Microscopy

In course

Description

This lecture covers the principles and applications of Total Internal Reflection Fluorescence Microscopy (TIRFM). It explains how TIRFM uses evanescent waves to selectively excite fluorophores near the coverslip surface, providing high-contrast images. The lecture discusses the limitations of conventional fluorescence microscopy and the conditions required for Total Internal Reflection (TIR). It also explores the concept of evanescent waves in TIR, the penetration depth of the evanescent wave, and various fluorescence microscopy techniques. Furthermore, it delves into the details of Fluorescence Recovery After Photobleaching (FRAP) method, its applications, kinetic analysis, and equipment. The lecture concludes with examples of FRAP experiments and the measurement of diffusion coefficients using FRAP.

Instructors (2)

Official source

https://mediaspace.epfl.ch/media/0_w3d1a33z

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Related concepts (54)

Diffuse reflection

Diffuse reflection is the reflection of light or other waves or particles from a surface such that a ray incident on the surface is scattered at many angles rather than at just one angle as in the case of specular reflection. An ideal diffuse reflecting surface is said to exhibit Lambertian reflection, meaning that there is equal luminance when viewed from all directions lying in the half-space adjacent to the surface.

Total internal reflection

In physics, total internal reflection (TIR) is the phenomenon in which waves arriving at the interface (boundary) from one medium to another (e.g., from water to air) are not refracted into the second ("external") medium, but completely reflected back into the first ("internal") medium. It occurs when the second medium has a higher wave speed (i.e., lower refractive index) than the first, and the waves are incident at a sufficiently oblique angle on the interface.

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.

Fresnel equations

The Fresnel equations (or Fresnel coefficients) describe the reflection and transmission of light (or electromagnetic radiation in general) when incident on an interface between different optical media. They were deduced by Augustin-Jean Fresnel (freɪˈnɛl) who was the first to understand that light is a transverse wave, even though no one realized that the "vibrations" of the wave were electric and magnetic fields.

Confocal microscopy

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.