In all forms of optical microscopy, the specimen scatters light through processes that include diffraction, refraction, reflection, and absorption. Transparent specimens imaged by phase contrast techniques diffract light that is retarded by one-quarter wavelength (90 degrees) with respect to undiffracted (surround) incident illumination, whereas opaque specimens, such as diffraction gratings, diffract light that is 180-degrees (one-half wavelength) out of phase with the surround illumination. This interactive tutorial explores diffraction of light by a periodic grating in a phase contrast microscope.

The tutorial initializes with a conoscopic view of the objective rear focal plane in a phase contrast microscope appearing in the window entitled: Diffraction Pattern. In this tutorial, the specimen is a variable opaque diffraction grating that gives rise to higher-order diffraction patterns, which can be observed in the objective rear focal plane. The central segmented circular white ring in the Diffraction Pattern window represents undiffracted light passing through the condenser annulus. Diffracted light, which is separated into a colorful spectrum according to wavelength (red, green, and blue), appears as successively higher (first and second) order circular rings to the left and right of the central annulus. To operate the tutorial, translate the Line Spacing slider to the right in order to decrease the spacing of the diffraction grating and alter the position of higher order diffracted light wavefronts. Note that as the diffraction grating line spacing is decreased, the diffracted light rings move away from the central annulus and become more diffuse.

Light wavefronts passing through a grating are diffracted according to the wavelength spectrum of the incident light beam and periodicity of the grating. Individual wavefronts diffracted by successive grating lines are emitted as concentric spherical wavelets that interfere both constructively and destructively because they are all derived from the same wavefront and are therefore in phase. Wavefronts passing through the grating slits that are parallel to the incident light wave are referred to as zero order (undiffracted or surround) or direct light. Diffracted higher-order wavefronts are inclined at an angle (q) according to the equation:

where l is the wavelength of the wavefront, P is the grating slit spacing and M is an integer termed the diffraction order (e.g., M = 0 for direct light, ±1 for first order diffracted light, etc.) of light waves deviated by the grating. The combination of diffraction and interference effects on the light wave passing through the periodic grating produces a diffraction spectrum (see the Diffraction Pattern window), which occurs in a symmetrical pattern on both sides of the zero order direct light wave.

The periodic diffraction grating can now be used to examine Ernst Abbe's theory of image formation in the phase contrast microscope. When the line grating is placed on a microscope stage and illuminated with a parallel beam of light that is restricted in size by the condenser annulus, both zero and higher order diffracted light rays enter the front lens of the objective. Direct light that passes through the grating unaltered is imaged in the center of the optical axis on objective rear focal plane as an image of the illuminated condenser annular diaphragm. First and higher order diffracted light rays enter the objective at an angle and are focused as spectral renditions of the condenser annulus on both sides of the central circular annular diaphragm pattern at the objective rear focal plane. A linear relationship exists between the position of the diffracted light beams and their corresponding points on the periodic grating.

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