PRINCIPLES OF COLOR PHASE CONTRAST 97 



color phase contrast image the surround appears neutral. A phenome- 

 non of phase microscopy is that the image of a particle is accompanied 

 by a halo of the opposite sign. In other words, the dark phase contrast 

 image of a particle is surrovmded by a bright band or halo which is 

 brighter than the image of the general background, and the bright phase 

 contrast image is surrounded by a dark halo which is darker than the 

 image of the general surround. The colored phase contrast image of a 

 particle is surrounded by a halo of a different color. In order that a 

 phase microscope produce a colored image of an object specimen when 

 the microscope and illuminating system have been set up according to 

 the procedure for Kohler illumination, the curve which describes as a 

 function of wavelength the relative phase difference which the diffraction 

 plate introduces between the undeviated and the deviated light must 

 show that either of the following two conditions exists. In the one, a 

 limited band of wavelengths forms either a bright-contrast or a dark- 

 contrast image of the object specimen, and the remaining wavelengths 

 of the spectrum contribute an ordinary, non-phase contrast image of the 

 object specimen. In the second, the requirement for bright contrast is 

 satisfied for one set of wavelengths, the requirement for dark contrast 

 is satisfied for another set of wavelengths, and an ordinary image also may 

 be superimposed l)y a third band of wavelengths, preferably very narrow, 

 which lies between those producing the bright- and dark-contrast images 

 of the object specimen. More complicated or less favorable designs 

 which give rise to a color phase contrast image will not be discussed 

 here. The wavelengths that interfere to form a dark-contrast image of a 

 particle and those that produce an ordinary image of the particle and its 

 surround contribute to the color of the halo. The color corresponding 

 to the wavelengths that interfere constructively to form a bright-contrast 

 image of the particle is absent in the halo. The halo is a phenomenon 

 associated with the discontinuity in optical path due to the presence 

 of the object specimen, and the halo is not being taken into account here 

 when references are made to the image of the surround. Color phase 

 microscopy not only produces striking images, but it also helps the 

 microscopist to distinguish between those effects caused by small amounts 

 of absorption in the object specimen and those caused by small differences 

 in optical path. 



To achieve color phase microscopy by the Zernike method, the dis- 

 persion of the substance forming the conjugate area is so chosen in rela- 

 tion to the dispersion of the substance forming the complementary area 

 that phase contrast is not observed if the specimen is illuminated by some 

 wavelengths in the visible range but that good phase contrast exists if 

 the specimen is illuminated by other wavelengths. The two kinds of 



