A novel technique is presented for surface compensation and topography measurement of a specimen in fluid medium by digital holographic microscopy (DHM). In the measurement, the specimen is preserved in a culture dish full of liquid culture medium and an environmental vibration induces a series of ripples to create a nonuniform background on the reconstructed phase image. A background surface compensation algorithm is proposed to account for this problem. First, we distinguish the cell image from the non-uniform background and a morphological image operation is used to reduce the noise effect on the background surface areas. Then, an adaptive sampling from the background surface is employed, taking dense samples from the high-variation area while leaving the smooth region mostly untouched. A surface fitting algorithm based on the optimal bi-cubic functional approximation is used to establish a whole background surface for the phase image. Once the background surface is found, the background compensated phase can be obtained by subtracting the estimated background from the original phase image. From the experimental results, the proposed algorithm performs effectively in removing the non-uniform background of the phase image and has the ability to obtain the specimen topography inside fluid medium under environmental vibrations.
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