TIRM is a technique used to look at fluorescence from a sample located within the first few hundred nanometers of the surface (Figure 1). There are several good reviews that describe this method (e.g. Axelrod, 1989, 2001; Tokunaga et al., 1997; Ambrose et al., 1999). Here, we briefly describe the TIR method and its application to DNA sequencing. When light strikes an interface going from a high refractive index medium to a low refractive index medium at an angle greater than the critical angle 0c, it undergoes a total internal reflection. The critical angle is given by Snell's law:
where nj(2) is the refractive index of the first (second) medium, and n1 > n2. In the lower refractive index medium, there is an exponentially decaying electromagnetic field called the ''evanescent wave''. The evanescent wave excites fluorescent molecules within about 150 nm of the surface, and its intensity at the surface can be higher than the intensity of the incident beam (Ambrose et al, 1999).
The fluorescence from the surface-bound molecules that are illuminated by the evanescent field, is detected by a microscope objective, through fluorescence filters by high sensitivity cooled CCD cameras. As only the vicinity of the surface is illuminated, there is a dramatic reduction of the noise from the bulk fluids, and surface-bound single molecules can be monitored with high signal to noise (Yildiz et al., 2003).
u n2: low refractive index a
fluorophores u n2: low refractive index fluorophores
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