In apodized phase contrast microscopy, halo attenuation and an increase in specimen contrast can be obtained by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density filter thin films applied to the phase plate surrounding the phase film as illustrated in the tutorial window.
llumination from the light source passes through a hypothetical spherically shaped specimen (phase object), whose size is adjustable with the Specimen Size slider. The tutorial initializes with the specimen set to the smallest size (slider bar on the left-hand side) by the slider. The speed of the tutorial can be adjusted using the Applet Speed slider.
As the thickness of the specimen is increased by translating the slider to the right, the phase difference between the specimen and the surrounding medium increases and the angle of light diffracted by the specimen becomes smaller. The diffracted light passes through an apodized phase plate (located in the rear focal plane of the objective), which contains two neutral density filter rings (having a 50-percent transmittance value) surrounding the phase ring. The width of the apodization neutral density filter rings is based on the diffraction angle according to the calculation:
Where θ is the angle of diffraction and δ is the phase difference between the specimen and the surrounding medium. Contrast is controlled by the transmittance of both the apodized (50 percent transmittance) and the dark low (DL) phase (25 percent transmittance) rings. Most of the low-spatial-frequency light diffracted by large specimens passes through these phase rings, while much of the light diffracted by small specimens (having a much higher spatial frequency) occurs at larger angles and passes through transparent portions of the phase plate. Light passing through the phase plates is attenuated and is represented by a decrease in color saturation for the arrows and waves in this tutorial.
Tatsuro Otaki - Optical Design Department, Instruments Company, Nikon Corporation, 1-6-3 Nishi-Ohi, Shinagawa-ku, Tokyo, 140-8601, Japan.
Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.