Digital holographic phase imaging of particles embedded in microscopic structures in three dimensions

Abstract

We present a three-dimensional microscopic technique based on digital holographic imaging, which allows highly accurate axial localization of features inside of a three dimensional sample. When a light wave is propagating through, or reflecting from, a microscopic object, the phase changes can be converted into intensity variations using the existing digital microscopic techniques. The phase change indicates the change in the optical path length, which can be then converted to physical thickness, providing the sample height information. This property of holograms is used in phasecontrast techniques, and can also be used for quantitative 3D imaging. However, if the sample contains features with different indices of refraction, this method can only provide the overall optical thickness, and cannot determine where in the axial direction the particular feature is located. As a result, the application of Digital Holographic Microscopy to imaging of organelles within live cells, or defects within semiconductor substrates, is limited to overall morphology of the sample. To determine the axial location of features inside of a three dimensional sample, we developed a phase image processing method based on analyzing images taken from non-zero incident angles. When compared, these images can discriminate between various axial depths of features, while still retaining the information about the overall thickness profile of the sample.