Multiple wavelength or multiple shear distance quantitative differential interference contrast microscopy

Abstract

A differential interference contrast (DIC) microscope system is provided comprising: (a) an illumination source for illuminating a sample; (b) a lens system for viewing the illuminated sample, including an objective, defining an optical axis; (c) a DIC prism having a shear angle for separating an input light beam into two, orthogonally polarized output beams that are separated by the shear angle; (d) at least one detector system for receiving a sample image; (e) a mechanism for modulating the phase of the DIC image; (f) a mechanism for selecting the wavelength of light used in acquiring at least two sets of DIC data taken in a single shear direction; and (g) a mechanism for manipulating the multiple DIC data sets to synthesize data that appear to have been taken at a longer synthetic wavelength. The various approaches disclosed and claimed herein permit the measurement of surfaces with a greater slope than is possible with a single wavelength or shear distance. The methods disclosed herein are applicable to measurement of slope in a single direction or in two directions. The disclosed methods are also compatible with the methods and devices of wavelength multiplexed quantitative DIC microscopy for obtaining slope data in two directions. An additional benefit of the disclosed techniques is the retention of the measurement sensitivity of a shorter wavelength or longer shear distance while increasing the measurement range corresponding to the long, effective wavelength obtained. However, the measurements are still limited to within the depth of focus of the microscope.