5° 



FUNDAMENTALS OF SUBM I C RO SC OPI C MORPHOLOGY 



Carrying out the same experiment with a dibasic acid, the film 

 thickness found is half that of the corresponding monobasic acid, 

 the surface occupied being twice as large. For example, the molecular 

 surface of nonyl acid CH3(CH2)7COOH is 25 A^, while sebacic acid 

 COOH(CH2)8COOH fills an area of 57 A- (Meyer and Mark, 1930). 

 This can be explained by assuming that both the carboxyl groups of 

 the dicarbon acid are dipping into the water, which means that the 

 molecule is bent (Fig. 43d). Such bending is made possible by the free 

 rotation around C-C-bonds. 



When a slide is dipped into the liquid on which a molecular monolayer is 

 spread, and then withdrawn, it is coated by a double layer of that compound. 

 If this procedure is repeated, two, three, four and more double layers may 

 be deposited on the glass slide. Such experiments can be performed with 

 stearate films whose double layers measure 48.8 A; so a slide can be coated 

 in stages with layers of any multiple of 48.8 A. 



Preparations hke these can be used for the determination of the sub- 

 microscopic thickness of very thin objects, provided they have a similar 

 refractive index to the stearate film for comparison. This method is based 

 on the fact that the intensity of the light reflected from a glass surface 

 diminishes when it is covered by a thin transparent film. The variables 

 involved in this phenomenon are the refractive indices of film, supporting 

 material and medium (usually air) through which they are viewed, and the 

 thickness of the film. The reflectivity depends further on the angle of inci- 

 dence and the wavelength of the light; both are kept constant by using an 

 appropriate vertical illumination. In a comparison microscope, called a /(?/)/(?- 

 scope (Waugh, 1950), the density of the biological object, e.g. ghosts of 

 erythrocytes, can be compared with the density of stearate films of known 

 thickness. Before a measurement is possible, the refractive indices of the 

 object and the comparison film must be determined, because they must be 

 ahke. This is done by using a set of glass sUdes covering a range of re- 

 fractive indices in small increments. The slide on which the object shows 

 the same reflectivity as the clean glass indicates its refractive index. 

 Mixtures of barium stearate and stearic acid are used to adjust the index 

 of the stearate film to that of the object. The eftect of reducing the intensity 

 of the reflected light is greatest when there is considerable disparity between 

 the refractivity of the support and that of the object; hence, the greater the 

 difl"erence in refractive index between object/film and glass, the better is 

 the determination of the thicls:ness of the object. 



With this method Waugh (1950) has found that the thickness of the mem- 

 brane in the red blood cells of the rabbit is 2 1 5 A ± 1 5 A at p^ 6 (cf. p. 264). 



Although proteins are to a certain extent hydrophilic, they, too, 

 form surface films. Ovalbumin, for instance, spreads on the surface of 



