POLARIZATION OPTICAL METHOD 277 POLARIZATION OPTICAL METHOD 



and Larsen (J. Nat. Cancer Inst., 1951, 

 11, 1187-1222) have shown that in Strain 

 A mice, adenomata may arise from them 

 during continuous administration of 

 urethane, 0.1% in the drinking water. 

 Macklin (J. Thor. Surg., 1938, 7, 536- 

 551; Trans. Roy. Soc. of Can., 1946, 

 Sect. V, 40, 93-111; and BioL BulL, 

 1949, 96, 173-178) has admitted a high 

 degree of mitotic potency in the pneu- 

 monocyte (epicyte) and has accepted 

 it as a primary center for lung cancer. 

 Pneumonocytes may become phago- 

 c>i;ic and appear with more or less 

 foreign material within the cytoplasm. 

 When such cells become free they take 

 a rounded or oval form and are known 

 as epithelial phagocytes, alveolar 

 phagocytes or Dust Cells (which see), 

 and are quite different from the histo- 

 cytes of the pulmonary connective 

 tissues. Free pneumonocytes without 

 particulate matter within them are 

 known as Foam Cells (which see). In 

 silver-wash preparations (see Silver 

 Lineation) these alveolar mural cells 

 are encircled each by a heavy golden- 

 brown line, and the air-faces are sprin- 

 kled with particles of the same hue. 



Large rounded granules of different 

 sizes are seen in them in frozen sections 

 from Regaud-fixed lungs which have 

 been well mordanted with potassium 

 bichromate and strongly stained with 

 Heidenhain's iron hematoxylin (Mack- 

 lin, C. C, Can. J. Res., D, 1950, 28, 5-15). 

 Similar granules, but gray or black, may 

 be seen in Aquax (which see) sections 

 from lungs that have been filled while 

 fresh with fixative containing from jg 

 to 1% of osmium tetroxide. Similar 

 granules, but of dark brown color, have 

 been described in them by Sjostrand and 

 Sjostrand (Zeitsch. f. mik.-anat. Forsch., 

 1938, 44, 370-411) where the living cells 

 have come into contact with blood and 

 an aldehyde, as pure formalin. They 

 find that this colored substance has 

 properties of hemin. 

 Polarization Optical Method. — Written by 

 Francis O. Schmitt, Dept. of Biology, 

 Massachusetts Institute of Technology, 

 Cambridge, Mass., May 19, 1950.— 

 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, Frey-Wyssling and 

 Schmitt listed at the end of this sec- 

 tion. 



The polarizing microscope is 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, iV, and No- 

 It is usually determined by the use of 

 a compensator which measures the 

 phase difference expressed as fractional 

 wavelength, 0, or retardation, r, ex- 

 pressed in ra/i. Thickness of the speci- 

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



birefringence = N, — No = -y = -3. 



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 diag- 

 nosing the ultrastructure of biological 

 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 



