Subnanometric measurements of evanescent wave penetration depth using total internal reflection microscopy combined with fluorescent correlation spectroscopy

In total internal reflection microscopy (TIRM), quantitative interpretation of results often requires a precise knowledge of the penetration depth of the evanescent wave. Standard TIRM practice is to calculate this depth from the microscope’s geometry, but this can introduce significant errors. We show how to calibrate the penetration depth of an evanescent wave in TIRM. An evanescent wave is obtained by illuminating a surface at an incident angle greater than the critical angle. Its penetration depth generally depends on the wavelength and the incident angle of the illumination, and on the indices of refraction on either side of the reflecting suface, but cannot be larger than the field of view. By introducing a fluorescent species (such as fluorescein) and measuring its diffusion time, it is possible to measure very precisely the penetration depth of the evanescent wave.