PLEUROPNEUMONIA 



197 



POLiVRIZATION OPTICAL 



W., Austral. J. Exp. BioL & Med. Sci., 

 1935,13,149-155). 



Plehn's Stain for malaria plasmodia is de- 

 scribed by Craig, p. 289 as uncertain in 

 its action and is not recommended if 

 other modifications of Romanowsky 

 stain are available. 



Plimmer's Bodies, see Bird's Eye Inclu- 

 sions. 



Polarization Optical Method. — Written by 

 Francis O. Schmitt, Dept. of Biology, 

 Massachusetts Institute of Technology, 

 Cambridge, Mass., May 28, 1946.— 

 The examination of tissues and cells 

 with the polarizing microscope gives 

 information about the presence of 

 preferentially oriented constituents, 

 the direction of their orientation, their 

 shape, regularity of internal construc- 

 tion, partial volume and refractive in- 

 dex. Details of the theory and methods 

 by which such information may be ob- 

 tained are contained in the books and 

 papers of Schmidt (1), Frey-Wyssling 

 (2) and Schmitt (3, 4). 



The polarizing microscope 's equipped 

 with a polarizer (nicol prism or polaroid 

 disc) below the condenser and an ana- 

 lyzer in the draw tube above the objec- 

 tive. Between the analyzer and objec- 

 tive is a slot into which may be inserted 

 a compensator or gypsum plate. When 

 the planes of polarization of polarizer 

 and analyzer are perpendicular no light 

 passes through the ocular. If a speci- 

 men is now placed on the stage, oriented 

 constituents may become visible on a 

 dark field. The intensity will be maxi- 

 mum when the distinguishing direction 

 of the object, such as a fiber, is oriented 

 at 45^ to the planes of polarization of 

 polarizer and analyzer. Objects hav- 

 ing internal regularity of structure may 

 have two descriptive refractive indices, 

 hence show double refraction or bire- 

 fringence. It is the object of polarized 

 light microscopy to detect, measure and 

 interpret this birefringence. 



Birefringence is numerically equal 

 to the difference between the two de- 

 scriptive refractive indices, A^e and A^o- 

 It is usually determined by the use of 

 a compensator which measures the 

 phase difference expressed as fractional 

 wavelength, 6, or retardation, r, ex- 

 pressed in mju. Thickness of the speci- 

 men, d, is also expressed in m/x. Then 



u- i- • ^' AT o\ r 



birefringence = rMo — No = -t-=j. 



Commonly used are the Berek, quar- 

 ter-wave (S^narmont) and Kohler ro- 

 tating mica-plate compensators, in or- 

 der of increasing sensitivity. 



Besides the magnitude of birefring- 

 ence its sign is of importance in diagf 

 nosing the ultrastructure of biologica- 



constituents. If the refractive index 

 for vibrations paralleling the distinc- 

 tive direction, e.g. the long axis of a 

 fiber, is greater than that for vibrations 

 perpendicular to this direction, the 

 birefringence is positive with respect to 

 this direction. If the refractive index 

 relations are reversed the birefringence 

 is negative. Most protein and carbo- 

 hydrate fibers show positive birefring- 

 ence while nucleic acid and nucleo- 

 proteins usually show negative 

 birefringence. While the sign of bire- 

 fringence may be determined with 

 compensators, the gypsum Red I plate 

 may be very useful. When this plate 

 is inserted into the compensator slot, 

 the field appears red if the nicols are 

 crossed. Birefringent objects show 

 addition or subtraction colors, such as 

 blue or yellow, respectively, depending 

 on the orientation of the object with 

 respect to the planes of polarizer and 

 analyzer and on the sign of birefring- 

 ence. Thus a fiber of connective tissue 

 or muscle will appear blue in one diag- 

 onal position and yellow in the diagonal 

 perpendicular thereto; this is because 

 these fibers manifest birefringence 

 which is positive with respect to the 

 fiber axis. A nerve fiber shows the 

 same colors in its myelin sheath except 

 that the diagonal positions in which it 

 shows these colors are reversed from 

 those of the above case; this is because 

 the myelin sheath manifests birefrin- 

 gence v/hich is negative with respect to 

 the fiber axis. 



The birefringence of most biological 

 objects is due to regularity of structure 

 of components considerably smaller 

 than the wavelength of light. To get 

 at the nature of these components, one 

 studies the relation of the birefringence 

 to the refractive index of the medium 

 in which the object is immersed, using 

 consecutively a number of media (us- 

 ually organic solvents) of varying re- 

 fractive index. Application of Wie- 

 ner's theory then makes it possible to 

 deduce the orientation of the submicro- 

 scopic particles as well as their internal 

 regularity of structure, refractive in- 

 dices and approximate partial volumes. 



Electron microscope observations 

 have confirmed many of the deductions 

 based on the polarization optical anal- 

 ysis of tissue ultrastructure. This 

 method will continue to be of impor- 

 tance biologically despite the great 

 possibilities of the electron microscopy, 

 for the polarized light method is appli- 

 cable to tissues in the fresh state. See 

 Schmidt,W.J.,DicDoppelbrechungvon 

 Karyoplasma, Zytoplasma und Meta- 

 plasma, Berlin Geb. Borntrager, 1937. 



