XIII. ACTION SPECTRA AND ABSORPTION SPECTRA 429 



length of the incident light, hut there are complicating factors, such 

 as interference, which make this rule far from universal (see 1,10). 

 For particles with diameters near the wavelength of the incident 

 light, the Rayleigh formula is obeyed, according to which scattering 

 increases inversely with the fourth power of the wavelength. Scatter- 

 ing by larger particles such as are often encountered in living systems, 

 e.g., scattering by reflection and refraction at the boundaries of cells, 

 is usually dependent to a much less extent upon wavelength. 



Ordinary methods of al)sorption spectroscopy, like those described 

 above, succeed because the systems dealt with do not scatter appre- 

 ciably in the spectral regions studied. Scattering introduces error, 

 w^hen such methods are used, because the beam that enters the ab- 

 sorbing medium normal to its surface is scattered in all directions, 

 more or less at random, so that much of the light does not reach the 

 measuring element, which is arranged to receive an emergent beam 

 normal to its surface. When scattering is high, only a small fraction 

 of the light that has passed through the absorbing substance may 

 reach the measuring element, and hence the estimated absorption is 

 higher than the true absorption, since much of the transmitted light 

 is not measured. The amount of loss by scattering must depend upon 

 the geometric arrangement of the instrument, as well as upon the de- 

 gree of scattering by the material. Thus, measurements of absorp- 

 tion by scattering materials obtained with ordinary instruments usu- 

 ally have little significance. 



There are no standard methods for measuring absorption by scat- 

 tering materials, and their development is to be desired. Several de- 

 vices have been used. One is to diffuse the beam before it enters the 

 absorbing material by interposing a diffusing plate, e.g., a plate of 

 quartz with a ground surface, so that the entering light is already 

 completely scattei-ed. This method uses ordinary equipment, but 

 has the disadvantage that the intensity may be so much reduced 

 that very sensitive measuring devices must be employed. Essen- 

 tially the same result is accomplished in the microscope since the il- 

 lumination enters the material on the stage from a wide angle so that 

 this instrument is quite satisfactory for the determination of the ab- 

 sorption of various components of living cells. Another method is to 

 place the absorbing material very close to a large flat-surfaced receiv- 

 ing element, or one made in the form of an integrating sphere. Thus 

 the element receives most of the light scattered over 180° solid angle. 

 Kirby-Smith {20) developed a method for studying the absorption of 



