Differential Interference Contrast (Nomarski) Imaging

Differential interference contrast (Nomarski) imaging is a modification phase microscopy. Transparent structures may be visualized in phase microscopy by producing contrast from refractive index inhomogeneities in the sample rather than from light absorption inhomogeneities. When light passes through a sample which is made up of elements whose refractive index differ, the phase of the wavefront will be altered because of the varying optical path-lengths traversed (light travels slower in regions of higher refractive index). Contrast is obtained by converting these phase changes into amplitude changes. Phase microscopy is well suited to studies of living tissue as samples do not have to be fixed and stained in order for internal structure to become visible.

Nomarski imaging is a phase imaging technique that gives good rejection of out-of-focus interference. It provides an image of the gradient (i.e. spatial rate of change) of refractive index inhomogeneities in the sample. The technique essentially acts as a high-pass filter that emphasizes edges and lines. Out-of-focus refractive index changes will be blurred and have a shallow spatial gradient in the focal plane, they will therefore not contribute much to the contrast of the image. Nomarski microscopy is generally much more benign to living specimens than fluorescence microscopy which can produce phototoxic effects. However, as Nomarski imaging produces contrast from refractive index gradients, there is very limited scope for developing labeling techniques that can reveal specific components of a specimen.