In this regard, the Nomarski prism and objective serve an identical function for incoming light waves as the first prism and condenser optical system in a transmitted light microscope. The shear produced when the light waves pass through the prism on the way to the objective is cancelled during their second journey through the prism upon returning from the specimen surface. Light passes through the same Nomarski prism twice, traveling in opposite directions, with reflected light DIC. Formation of the final image in differential interference contrast microscopy is the result of interference between two distinct wavefronts that reach the image plane slightly out of phase with each other, and is not a simple algebraic summation of intensities reflected toward the image plane, as is the case with other imaging modes.Ī significant difference between differential interference contrast in transmitted and reflected light microscopy is that two Nomarski (or Wollaston) prisms are required for beam shearing and recombination in the former technique, whereas only a single prism is necessary in the reflected light configuration. Components of the orthogonal wavefronts that are parallel to the analyzer transmission vector are able to pass through in a common azimuth, and subsequently undergo interference in the plane of the eyepiece fixed diaphragm to generate amplitude fluctuations and form the DIC image. After exiting the Nomarski prism, the wavefronts pass through the half-mirror on a straight trajectory, and then encounter the analyzer (a second polarizer) positioned with the transmission axis oriented in a North-South direction. Reflected wavefronts, which experience varying optical path differences as a function of specimen surface topography, are gathered by the objective and focused on the interference plane of the Nomarski prism where they are recombined to eliminate shear. The deflected light waves, which are now traveling along the microscope optical axis, enter a Nomarski prism housed above the objective in the microscope nosepiece where they are separated into polarized orthogonal components and sheared according to the geometry of the birefringent prism.Īcting in the capacity of a high numerical aperture, perfectly aligned, and optically corrected illumination condenser, the microscope objective focuses sheared orthogonal wavefronts produced by the Nomarski prism onto the surface of an opaque specimen. Linearly polarized light exiting the polarizer is reflected from the surface of a half-mirror placed at a 45-degree angle to the incident beam. Illumination generated by the light source passes through the aperture and field diaphragms (not illustrated) in a vertical (episcopic) illuminator before encountering a linear polarizer positioned with the transmission axis oriented East-West with respect to the microscope frame. Unlike the situation with transmitted light and semi-transparent phase specimens, the image created in reflected light DIC can often be interpreted as a true three-dimensional representation of the surface geometry, provided a clear distinction can be realized between raised and lowered regions in the specimen.Ī schematic cutaway diagram of the key optical train components in a reflected light differential interference contrast microscope is presented in Figure 1. Slopes, valleys, and other discontinuities on the surface of the specimen create optical path differences, which are transformed by reflected light DIC microscopy into amplitude or intensity variations that reveal a topographical profile.
Reflected light microscopy is one of the most common techniques applied in the examination of opaque specimens that are usually highly reflective and, therefore, do not absorb or transmit a significant amount of the incident light.
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