Total Internal Reflection Microscopy: Distortion Caused by Additive Noise

Abstract

Total internal reflection microscopy (TIRM) is an optical technique for monitoring Brownian fluctuations in the separation between a single microscopic sphere and a flat plate in aqueous media. The sphere is levitated above the plate by colloidal forces such as double-layer or steric repulsion. Changes in elevation as small as 1 nm can be detected by measuring the light scattered by a single sphere when illuminated by an evanescent wave. From the Boltzmann distribution of elevations sampled by the sphere over a long time, the potential energy (PE) profile can be determined with a resolution of about 0.1kT. By corrupting clean data (having a mean scattering intensity of Īs and a standard deviation of σs) obtained by Brownian dynamics simulations with various levels of additive background noise (having a mean background intensity of Īb and a standard deviation of σb), we show how the PE profiles obtained from TIRM are distorted. Increasing Īb narrows the profile and shifts it toward smaller elevations; conv...