MICROSPECTROPHOTOMETRY 



208 



MICROSPECTROPHOTOMETRY 



a total path length considerably longer 

 than the dimensions of the object. 

 This effect will enhance the absorption 

 of the object and may be variable with 

 wavelength. 4) The relationship be- 

 tween the optical density of an object 

 and the concentration of the specific 

 substance must be demonstrated since 

 it cannot be assumed that the linearity 

 predicted by Beer's Law will hold. 

 5) The effect of inhomogeneous distri- 

 bution of the absorbing material in the 

 planes perpendicular to the optical 

 axis must be estimated since relatively 

 small departures from homogeneity 

 will lead to significant alterations in the 

 absorption due to a given amount of 

 material. 



If the absolute quantity of a given 

 substance in an intracellular object is 

 to be determined, two further condi- 

 tions must be met. 6) The specific 

 extinction of the substance in question, 

 that is, the absorption per mole per 

 liter must be known. 7) The influences 

 of the conditions noted above on the 

 extinction must be determined quan- 

 titatively. If accepted analytical prac- 

 tices are to be followed, this would mean 

 that the absorption due to a known 

 amount of the specific substance when 

 added to the intracellular object be 

 determined or that the amount of the 

 substance found in the object by inde- 

 pendent analytical means be correlated 

 with its absorption characteristics. 



Unfortunately, presently available 

 techniques can meet the above condi- 

 tions only in part. The procedures 

 thus far worked out or suggested are 

 noted below. 



/ . The problem of complex composition 

 of the object. If it can be shown that 

 the composition of the object is uniform 

 throughout its optical depth, then the 

 relative contribution of individual sub- 

 stances to the overall absorption spec- 

 trum may be worked out by comparison 

 of the latter with individually deter- 

 mined spectra of the separate compo- 

 nents. However, large differences in the 

 height of the maxima frequently make 

 this procedure a difficult one. To 

 demonstrate that this optical uni- 

 formity exists, it must be shown that 

 the absorption spectrum of the object 

 is constant for all thickness (obtained, 

 for example, by sectioning). Where 

 this is not possible as in the case of 

 structures enclosed within intact liv- 

 ing cells an optical method may be used 

 (Commoner, B., Discussions of the 

 Faraday Society, 1950, No. 9, 449-460). 

 This procedure may be applied to ob- 

 jects in which one component is dis- 

 tributed in an invariant layer in a cell 



which varies considerably in thickness. 

 In this case it is possible to determine 

 the absorption due to the separate com- 

 ponents in situ by measuring the ab- 

 sorption spectra of two regions in the 

 cell which differ in thickness by a known 

 amount. In favorable instances this 

 method may be used to determine the 

 absorption spectrum of the nucleus con- 

 tained within a living cell. Thus far 

 the procedure has been applied only to 

 certain types of plant cells. Failure 

 to conform with this condition casts 

 doubt on the meaning ascribed to the 

 absorption spectra of structures such 

 as the nucleolus which have been ob- 

 tained without detailed analysis of the 

 contribution made by over-lying and 

 under-lying material. 



2. Non-specific light losses. It has 

 been frequently assumed (Caspersson, 

 T., Cell growth and cell function. New 

 York: Norton, 1949) that light lost 

 due to scattering is related to wave- 

 length according to the Rayleigh equa- 

 tion. Using this assumption and the 

 further assumption that at some spe- 

 cific wavelength range such as 300-350 

 mM no specific absorption occurs, the 

 scattering losses are calculated by ex- 

 trapolation from the readings obtained 

 in this limited range. The usefulness 

 of this method is considerably weakened 

 by the fact that the first assumption 

 has never been demonstrated to be true 

 for intracellular objects and that the 

 second assumption can be true only very 

 rarely. An experimental determina- 

 tion of losses due to scattering may be 

 made by an apparatus described by 

 Caspersson, T. (Cell growth and cell 

 function, New York: Norton, 1949) 

 which measures the light emerging 

 from the object at various angles from 

 the optical axis. Scattering losses 

 may vary considerably with the physi- 

 cal state of the object and are, there- 

 fore, very sensitive to fixation proce- 

 dure, etc. 



3. The length of the optical path. 

 Thus far no method for determining this 

 value has been proposed and in prac- 

 tice the only approach to the value of 

 this dimension is the observed thickness 

 of the object. Thus in making this 

 assumption, calculations are exposed to 

 an error of unknown magnitude. 



4- The validity of Beer's law. This 

 essential determination has thus far 

 been carried out in only one instance 

 (Commoner, B., Discussions of the 

 Faraday Society, 1950, No. 9, 449-460) 

 in which the cellular material occurred 

 in solution in the vacuole of a mature 

 plant cell. In this case it was possible 

 to alter the concentration of the dis- 



