SECTION III. SPECTROGRAPHIE ULTRA- VIOLETTE 



ULTRA VIOLETTMIKROSPEKTROPHOTOMETRIE 



W. SANDRITTER* 



Senckenbergisches Pathologisches Institut der Universitat 



Frank furt am Main 



The cytophotoraetry with UV-light as introduced by Caspersson in 1936 

 placed a new sphère of investigational possibilities within reach of 

 photometric methods. The measurements of biological objects with dimen- 

 sions of a few u and concentrations and weights in the range of 10' g 

 became a practical reality. A review of this complex field, in which many 

 of the fundamentals are still being discussed, should be directed toward, 

 1) revealing possible sources of error 2) the comparison of UV-cytophoto- 

 metry proven methods of measurement currently in use 3) emphasizing 

 those areas in the médical and biological sciences where the new method 

 may be profitably applied. 



The first part, using our own equipment as examples, describes the 

 apparatus for photography with UV-light. A one beam cytophotometer 

 which was built from purchasable éléments and a two-beam scanning- 

 cytophotometer with automatic registration are also described. The 

 discussion of theoretical fundamentals gives spécial attention ^o the 

 possibilities of errors introduced both by the apparatus and the biological 

 object. Nine basic conditions which are prerequisites for cytophotometric 

 measurements are defined. 



The numerical aperture of the objective must be not less than 0*85 to 

 insure inclusion of stray light. Smaller numerical apertures lead to an 

 increase of extinction. The numerical aperture of the condensor must be 

 smaller than that of the objective {%-%). The smallest measurable object 

 is approximatly 3 to 4 times larger than the wave îength of the incoming 



light. ... , 



The non-homogenous character of absorbing material (in interdepen- ■ 



dence on the distribution of chromophores and the peak of extinction) 



leads, by large photomultiplier openings, to a lowering of the measured 



extinction. This error can be eliminated by using a small photomultiplier 



opening. The various indexes of refraction within the biological object 



itself increase the stray light emd consequently the non-absorption light 



loss. Freezing drying of the tissue and matching of the refraction indexes 



are prerequisites for accurate measurements. 



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