Total Internal Reflection Microscopy (TIRM)

Abstract

Total internal reflection microscopy (TIRM) uses the evanescent wave field (Section 1.1.1) as an illumination light source and allows near-field characterization within the penetration depth z _p of less than 1 (m of the evanescent wave field:

                         $z_{p}=\frac{\lambda_0}{4_\pi}(n_i^2 sin^2 \theta_i-n_t^2)^{-{\frac{1}{2}}}$      (1.2)

The fundamental concept of TIRM is simple, requiring an excitation light beam traveling at a high incident angle to create the evanescent wave field [55,56]. Such a narrowly defined illumination depth is considered to be a highly effective way of overcoming the background noise that is often the biggest problem in single molecule imaging [57]. The principles and configuration of TIRM are summarized in Section 2.1 and the ratiometric TIRM imaging analysis technique is presented in Section 2.2. Finally, selected near-field applications of TIRM are discussed in Section 2.3.