Orientation-Independent DIC Microscopy with Fast Switching Shear Direction and its Applications

Abstract

A conventional DIC (differential interference contrast) microscope shows the two-dimensional distribution of refractive index gradient encountered along the shear direction. Thus contrast of DIC images varies proportionally with the cosine of the angle made by the azimuth of the refractive index gradient and the direction of wavefront shear. It is therefore necessary to examine unknown objects at several azimuth orientations. To overcome the limitations of available systems, we have built an assembly, which allows the bias and shear directions to be switched rapidly without mechanically rotating the specimen or the prisms. The assembly consists of two standard Nomarski prisms with liquid crystal polarization rotator in between. When the polarization rotator is in OFF state, the total shear direction of the assembly is at +45-deg to the shear direction of the first prism. If the polarization rotator is in ON state, the total shear direction is at −45-deg to the shear direction of the first prism. Thus, when one would switch the polarization, the shear direction would be rotated by 90-deg. We added one assembly to the illumination path and another one to the imaging path of the standard Olympus BX-61 microscope. Also one variable liquid crystal retarder was installed in order to change a bias. Four raw DIC images at two orthogonal shear directions and two inverse biases are captured and processed within a second. Then the quantitative image of refractive index distribution within a thin optical section is displayed on a computer screen. The orientation-independent DIC data obtained can also be used to compute the quantitative distribution of refractive index gradient or to generate enhanced, regular DIC images with any desired shear direction. The OI-DIC system can be combined with other techniques, such as fluorescence and polarization microscopy.