Effect of Photon Counting Shot Noise on Total Internal Reflection Microscopy

Abstract

Total internal reflection microscopy (TIRM) measures changes in the distance between a colloidal particle and a transparent substrate by measuring the intensity of light scattered by the particle when it is illuminated by the evanescent field that is created from light totally internally reflected at the substrate interface. From these measurements, the height-dependent effective potential $\ddot{I}†(z)$ between the colloidal particle and the substrate can be measured. The spatial resolution with which TIRM can resolve the height $z$ and effective potential $\ddot{I}†(z)$ is limited by the intrinsic shot noise of the photon counting process used to measure the scattered light intensity. We develop a model to determine the spatial resolution with which TIRM can measure $\ddot{I}†(z)$ and verify its validity with simulations and experiments. We further establish the critical role of photon-counting statistics and the intensity integration time $\ddot{I}„$ in TIRM measurements, which is a trade-off between narrowing the width of the photon counting distribution and capturing the instantaneous position of the probe particle.

10 pages, 7 figures