128 INSTRUxMENTATION 



5. SOME EFFECTS OF VARYING THE DIMENSIONS AND OPTICAL 

 CHARACTERISTICS OF THE CONJUGATE AREA 



The fact has been emphasized that a compromise decision must be 

 made in choosing the dimensions of the conjugate area of the diffraction 

 plate designed for general use. The elementary theory was developed 

 in Chapter II to show the interdependence of the optical properties of 

 the particle and those of the diffraction plate when the problem of 

 optimum -contrast is being considered. In Section 1 of this chapter this 

 elementary theory was applied in an attempt to predict the range 

 of usefulness of a diffraction plate with an optical path step equal to 

 X/4 between the complementary and the conjugate areas and with a 

 lower energy transmission in the conjugate area than in the comple- 

 mentary area. The simple theory does not take into account either the 

 shape of the object specimen or the area occupied by it, and this theory 

 also does not include the effect on contrast of the size and location of the 

 conjugate area of the diffraction plate. 



In the previous general discussions statements have been made con- 

 cerning whether a particle appears brighter or darker than its surround. 

 However, observations with the phase microscope show that, when the 

 area occupied by a particle of uniform optical path becomes large, the 

 brightness at the center of the image of the particle is equal to that of 

 the image of a comparatively remote region of the surround, and only the 

 region near the boundary in the image of the particle appears darker or 

 brighter according to the type of contrast being produced. Further- 

 more, it is also seen that the image of a particle is surrounded by a halo, 

 A dark-contrast image is surrounded by a bright halo, and a bright- 

 contrast image is surrounded by a dark halo. This halo is an artifact. 

 Thus it is seen that phase contrast occurs only in the neighborhood of a 

 rapid change in the optical properties of a particle relative to its sur- 

 round. In order to achieve phase microscopy, the diffracted rays must 

 be deviated sufficiently relative to the undeviated rays so that the 

 deviated and undeviated bundles become fairly well separated at some 

 plane within the microscope if a diaphragm is introduced below or 

 within the substage condenser. The relative phase change introduced 

 between the deviated and the undeviated light by the occurrence of 

 diffraction lies in the neighborhood of 90° when the optical path differ- 

 ence between a transparent particle and its surround is a small fraction 

 of a wavelength. The method of phase microscopy is not applicable for 

 observing slow, gradual changes in optical path; in such cases interfer- 

 ometric means must be used. It is known, for example, that, when a 

 particle is slightly smaller than the limit of resolution of an objective, 

 the amount of light in the diffraction pattern diminishes rapidly with 



