A Phase-Shifting DIC Technique for Measuring 3D Phase Objects: Experimental Verification

Experimental verification of our previously proposed linear phase imaging technique for differential interference contrast microscopy (DIC) microscopy is presented. This technique first applies phase-shifting methods to DIC to acquire linear phase gradient images in two orthogonal directions. A special Fourier integration algorithm is then applied to the combined phase gradient images to create a single linear phase image in which intensity is proportional to phase. This overcomes the limitations of traditional DIC, which cannot accurately measure the phase (i.e. refractive index or thickness) of embedded 3D phase objects. The linear phase imaging technique is implemented using a standard DIC microscope altered to allow controlled phase shifting, a low noise CCD camera, and post-processing in Matlab. The results presented confirm the linear proportionality of intensity to phase in these images.