Three-dimensional single particle tracking using off-axis digital holographic microscopy

Abstract

Three dimensional particle tracking is useful technology to characterize live cell or surrounding environment by tracing small particles such as fluorescence beads or polystyrene beads which adhered to objective samples. In microscopy imaging system, the longitudinal(z axis) tracking of the particle is essential for implementation of three-dimensional particle tracking, however it's been still a challenging topic to find the exact position of the particle in z axis with high precision. In this study, we present that a novel technique to find the longitudinal position of the particle, as well as the transverse position(x,y axis) by applying the numerical reconstruction and focusing with digital holographic microscope. Transmission type off-axis digital holographic microscope is implemented for this experiment, based on Mach-Zehnder interferometer and 632.8nm HeNe laser is used as a coherent light source of the microscope and high-speed CMOS camera is utilized for acquiring the hologram. Digital holographic microscope makes it possible to record and reconstruct the phase and amplitude image of the sample. In order to find the position of the particle in z axis, we apply the numerical focusing algorithm, which enables the translation of the imaging focus without actual longitudinal movement of the sample. To demonstrate the presented method, Brownian movement of 3μm polystyrene sphere suspended in water is investigated in this experiment.