CHAPTER X 



Microspectroscopy and 

 Microspectrophotometry 



1. FUNDAMENTALS OF MICROSPECTROSCOPY 

 AND MICROSPECTROPHOTOMETRY 



In microscopy, investigating spectra in the same way as in chemical 

 analysis affords valuable information related to the constitution of 

 small samples. In histochemistry, for instance, it complements the 

 morphological aspect of the problems considered by determining 

 locahzed substances. 



Evolvement of reflecting objectives gave rise to the extension of 

 microspectroscopy. Complete absence of chromatism made them 

 particularly suitable in ultra-violet and infra-red applications as they 

 could be used without altering the focusing used in visible Hght: 

 obviously a very valuable and practical feature. Generally, micro- 

 spectrophotography determinations are spectral-absorption determina- 

 tions. The object, transparent to a lesser or greater degree, need not 

 be destroyed and its absorption is studied versus the wave-length. 

 Conventional spectrum analysis methods apply equally well to micro- 

 scopy and furnish either quahtative or quantitative analysis. 



In qualitative analysis, the spectrum of an area of the specimen 

 is used to seek the presence and define the nature of an unknown 

 substance. Then, the absorption curve of the spectrum is to be plotted 

 in order to determine the position and the structure of absorption 

 bands in a more or less extensive portion of the spectrum. 



In quantitative analysis, the purpose sought is to determine the 

 quantity of a substance whose presence is already ascertained. Two 

 procedures may be used: 



(a) The object is illuminated in monochromatic light; 



(b) The object is illuminated by a complex source and the spectral 

 analysis carried out after the light has passed through the object. 



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