Because excitation of fluorophores in the bulk of the specimen is avoided, confining the secondary fluorescence emission to a very thin region, a much higher signal-to-noise ratio is achieved compared to conventional widefield epifluorescence illumination. By comparison, this optical section thickness is approximately one-tenth that produced by confocal fluorescence microscopy techniques. Because of the exponential falloff of evanescent field intensity, the excitation of fluorophores is restricted to a region that is typically less than 100 nanometers in thickness. In a typical experimental setup, fluorophores located in the vicinity of the glass-liquid or plastic-liquid surface can be excited by the evanescent field, provided they have potential electronic transitions at energies within or very near the wavelength bandwidth of the illuminating beam. This evanescent field is identical in frequency to the incident light, and because it decays exponentially in intensity with distance from the interface, the field extends at most a few hundred nanometers into the specimen in the z direction (normal to the interface). Its refractive behavior is governed by Snell's Law : Formula 1 - Snell's LawĪlthough light no longer passes into the second medium when it is incident at angles greater than the critical angle, the reflected light generates a highly restricted electromagnetic field adjacent to the interface, in the lower-index medium. Total internal reflection is only possible in situations in which the propagating light encounters a boundary to a medium of lower refractive index. A collimated light beam propagating through one medium and reaching such an interface is either refracted as it enters the second medium, or reflected at the interface, depending upon the incident angle and the difference in refractive indices of the two media. In each case, refraction (or bending) of light as it encounters the interface between two media having different refractive indices ( n) results in confinement of a portion or all of the light to the higher-index medium. The physical phenomenon of total internal reflection ( TIR) has been relied upon in such seemingly diverse applications as modern fiber optic data transmission, and in the centuries-old utilization by diamond cutters to enhance the sparkle, or "fire", of cut gemstones. The availability of complete ready-to-use instrumentation systems for employment of the method, as well as developments in fluorophore technology, such as genetically encoded fluorescent species, have made it possible to investigate a number of cell membrane and other surface processes in a direct manner that was not previously possible. The concepts underlying TIRFM are not new, and much of the recent interest in, and enthusiasm for, the technique have come about due to technological advances that facilitate its use.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |