MICROSPECTROPHOTOMETRY 



207 



MICROSPECTROPHOTOMETRY 



Amer., 1950, 40, 283-290, Mellors, R. C, 

 Science, 1951, 112, 381-389. The prob- 

 lem of instrumental accuracy is dis- 

 cussed by Click, D., Engstrom, A. and 

 Malmstrom, B. G. (Science, 1951, 114, 

 253-258) and Caspersson, T. (Cell 

 growth and cell function, New York: 

 Norton, 1949). 



The light absorption due to the ob- 

 ject may be determined in the following 



ways. 



1. Direct method. The microscope 

 is focused on an area of the object 

 slide adjacent to the object itself. 

 The light impinging on the photo-cell 

 is then adjusted to give a fixed response 

 in the detector circuit. Without alter- 

 ing the optical conditions the slide is 

 then moved so that the object is now 

 centered in the field and a reading of 

 the detector response is taken. This 

 measurement compared with the first 

 one yields the per cent of the incident 

 light which is transmitted by the ob- 

 ject. 



2. Split-beam method. In this 

 method the light beam is split before 

 entering the microscope and a small 

 fraction directed toward an accessory 

 phototube and detector circuit. It is 

 then possible to evaluate the intensity 

 of the light reaching the main photo- 

 tube in terms of the intensity of the 

 beam directed toward the seconday 

 phototube. This method has the ad- 

 vantage of being independent of random 

 fluctuations in the intensity of the light 

 source. As in the direct method the 

 absorption of the object is determined 

 by comparison with a blank area in the 

 slide. 



Interpretation of data: The derivation 

 of valid conclusions from intracellular 

 absorption data is a considerably more 

 difficult task than the experimental 

 work itself; in fact, it is frequently the 

 case that data are obtained for which 

 there are as yet no valid interpretations. 

 The difficulties in interpretation arise 

 from the fact that little is known con- 

 cerning the physical state of intracel- 

 lular objects and its effect on their 

 optical properties. Since the rules for 

 analysis of absorption data are based 

 exclusively on the optical properties of 

 homogeneous systems such as gases and 

 dilute solutions, entirely new methods 

 need to be developed for the hetero- 

 geneous structures of the cell. 



For homogeneous solutions of ab- 

 sorbing materials, the following rela- 

 tions (the Beer-Lambert Laws) are 

 found, within limits, to hold: 



D = kcd 

 where c is the concentration of absorb- 

 ing material, d is the length of the opti- 



cal path through the absorbing layer, 

 and k is the extinction coefficient of the 

 substance in question. D, the optical 

 density, is defined by the expression 



D = logio y, where lo is the intensity 



of the incident beam and I the intensity 

 of the transmitted beam. The Beer- 

 Lambert relationship holds only where 

 all light absorption is due to the trap- 

 ping of photons by the substance in 

 question, and where each molecule in 

 the light path contributes equally to 

 the absorption. 



In microspectrophotometric work, 

 the value of D for a given optical sec- 

 tion through the object is determined 

 experimentally at various wavelengths. 

 This gives the absorption spectrum of 

 the entire object. Since the aim of the 

 measurement is usually the identifica- 

 tion and estimation of a specific sub- 

 stance present in the object, the initial 

 spectrum needs to be evaluated in the 

 light of the above relationships. 



In order to identify a specific sub- 

 stance from the absorption spectrum 

 of an object believed to contain this 

 substance, the following conditions 

 must be met. 1) It must be known 

 that the object contains no absorbing 

 material other than the substance in 

 question; or the relative contents and 

 absorption spectra of extraneous sub- 

 stances must be determined. Without 

 this information one cannot conclude 

 that a given absorption maximum in the 

 spectrum of the object is due to a spe- 

 cific substance ; such a maximum may re- 

 sult from the superposition of the 

 spectra of two or more different sub- 

 stances. 2) It must be known that the 

 absorption of light at any given wave- 

 length is due only to the trapping of 

 photons by the substance in question 

 and is not a consequence of scattering 

 and similar non-specific processes. In 

 general, light scattering rises with in- 

 crease in frequency but since sharp 

 changes in refractive index occur near 

 the absorption maximum of a sub- 

 stance, scattering effects may some- 

 times pass through a maximum with 

 variation in wavelength. 



If in addition to identification an 

 estimation of the relative arnount of 

 the substance present in the intracel- 

 lular object is to be made, the follow- 

 ing further conditions must be fulfilled. 

 3) The length of the optical path 

 through the object must be known. 

 This is not necessarily identical with 

 the thickness of the object for in hetero- 

 geneous systems internal reflections 

 may cause a significant fraction of the 

 entering beam to be reflected through 



