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Gradient images can be obtained using a rotating square mask to filter the angular spectra of the wavefront generated by a complex transmittance object. This method can be applied to measure the three-dimensional structure of microscopic biological samples through the relationship of the phase with the optical path length. This work describes the implementation of a system using an inverted optical microscope and shows the experimental results of phase maps generated by boar sperm cells.

Use of a Rotating Square Spatial-Frequency Filter to Map the Optical Path Length Variation in Microscopic Biological Samples