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Total Internal Reflection Fluorescence Microscope

The Principle of Total Internal Reflection

According to the principle of geometrical optics, when light in a medium with a higher refractive index reaches a boundary with a medium of lower refractive index, the wave will in general be partially reflected at the boundary surface, and partially refracted. With the increase of the angle of incidence, the intensity of the reflected light gradually increases and the intensity of the refracted light decreases. When the incident angle increases to angle 6c, the refraction angle is 90°. Then refraction of light passing the boundary between two media and the intensity closes to zero. When the incident angle 61 > 6c, refracted light no longer exists, the incident light is totally reflected. This phenomenon is called “total internal reflection” (TIR). When

Comparison of images acquired by 2-photon process with that by 1-photon process under multiple color conditions [42]

Fig. 2.4 Comparison of images acquired by 2-photon process with that by 1-photon process under multiple color conditions [42]

Fig. 2.5 Fluorophore excitation near surface by total internal reflection

refraction angle is 90°, the angle of incidence Qc called the “critical angle” or“total internal reflection angle.”

In the case of TIR, most of the light is reflected along the direction of the reflected light. Only a small portion of the reflected light propagates through a parallel interface of the surface and forms the electromagnetic field near the interface of the medium. This field is called the evanescent field, which decays exponentially with the increase of propagation distance, so in a very short distance (about a few hundred nanometers) the evanescent field will disappear. It is precisely that the energy can only excite the fluorescent probes close to the interface thanks to the nature of evanescent field, thus realizing the detection of single fluorescent molecule and researching on molecular interactions. This is very beneficial to the study of the characteristics of the cell membrane and other membrane structure.

 
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