Particle localization with total internal reflection illumination and differential detection

A theoretical framework of an alternative to TIRFM for high-speed reconstruction of the axial information from a fluorescent sample is presented. Two detectors are applied to capture the sample intensity, and the differential signal from the two detectors is used to determine the axial location with decreased detected noise. The axial response is analyzed theoretically; for greater accuracy, the distance dependence of the radiation patterns of samples near the interface is considered. Depth measurements with improved axial resolution and robustness to noise were obtained. The method may provide a reliable tool for 3-D reconstruction and tracking of fluorescent particles for studies of the mechanisms of cellular and subcellular processes near the cell surface.