MICROSPECTROPHOTOMETRY 



206 



MICROSPECTROPHOTOMETRY 



compared with ground substance. 

 Claude, A. Biological Symposia, 1943, 

 10, 111-129 estimates their size to be 

 50-300 ran and therefore beyond limits 

 of ordinary microscopic visibility. 

 These microsomes of Claude are ob- 

 viously not the ones which Hanstein 

 had in mind. According to Claude 

 they are essentially ribose nucleopro- 

 teins and phospholipins in definite 

 proportions. 

 Microspectrophotometry — Barry Commo- 

 ner, The Henry Shaw School of Botany, 

 Washington University, St. Louis 5. 

 November 28, 1951 — Microspectropho- 

 tometry is a technique for examination 

 of cells and cell structures designed to 

 yield data on the chemical composition 

 of these objects. The method is 

 based on the fact that given molecular 

 configurations absorb specific wave- 

 lengths of ultraviolet, visible or infra- 

 red radiation. Under ideal conditions, 

 as in dilute solutions, the molecular 

 group and therefore the substance in 

 which it occurs may be identified from 

 the shape of the absorption spectrum, 

 and its concentration in the sample 

 determined from the amount of absorp- 

 tion at a characteristic wavelength. 

 Unfortunately, cytological objects 

 never offer such simple, readily analyz- 

 able situations. Consequently, special 

 steps must be taken to evaluate the 

 absorption spectra of cytological 

 objects, and in most instances, data com- 

 parable with those obtained from solu- 

 tions are not yet attainable. Never- 

 theless, the technique has thus far 

 proved to be a valuable source of in- 

 formation on cell composition, and if 

 used with care can be advantageously 

 applied to a number of biological prob- 

 lems. 



The essential measurement in micro- 

 spectrophotometry is determination 

 of the reduction in intensity of a light 

 beam after passing through a cytologi- 

 cal object. This measurement, made 

 at a series of specific wavelengths, gives 

 the absorption spectrum of the object. 

 The measurements may be made by 

 passing a monochromatic beam through 

 the object; or by passing a heterogene- 

 ous beam through the object and then 

 dispersing it into a spectrum. Equip- 

 ment employing the second of these 

 methods has been described by (Mellors, 

 R. C, Science, 1951, 112, 381-389). 

 Since most microspectrophotometry 

 has employed the first principle of 

 operation, details for this type of ap- 

 paratus are given below. 



1. General. The fundamental parts 

 of a microspectrophotometer consist 

 of a suitable light source, a monochrom- 



ator, microscope optics, a photoelec- 

 tric tube which receives the projected 

 image of the object, and appropriate 

 means of measuring the phototube 

 response. 



2. Light sources. Ordinary automo- 

 bile headlight tungsten lamps operated 

 on a storage battery are suitable for 

 work in the visible spectrum. For work 

 in the ultraviolet ranges mercury dis- 

 charge tubes provide adequate light 

 intensity. High pressure mercury 

 lamps (such as General Electric AH6) 

 emit a continuous spectrum in the vis- 

 ible and ultraviolet superimposed on a 

 number of bright and dark lines. In 

 the infra-red ranges Nernst glowers are 

 suitable. 



3. The monochromator. The light 

 beam from source is directed into the 

 entrance slit of a monochromator, 

 thus giving an emergent beam of a 

 determined mean wavelength. The 

 monochromator should supply the con- 

 denser of the microscope with a beam 

 sufficient to fill the aperture of the 

 latter. 



4. The microscope. The optical ar- 

 rangement of the microscope must be 

 such as to give a true light image of the 

 object at the plane of the photocell. 

 The requirements for this condition 

 have been presented by Caspersson, 

 T. (Cell growth and cell function, New 

 York: Norton, 1949). For visible work 

 any good apochromatic system of suffi- 

 cient numerical aperture is adequate. 

 In the ultraviolet range quartz lenses 

 or reflecting objectives must be used. 

 The latter have the advantage of being 

 achromatic in the ultraviolet spectrum. 



5. The light detector. The most 

 suitable detector for this type of work 

 is the photomultiplier tube. The tube 

 is supplied with a suitable power source 

 and its output amplified and read off 

 a microammeter. Less conveniently 

 the direct output may be detected with 

 a galvanometer. 



The construction of microspectro- 

 photometric equipment has been de- 

 scribed in the following papers: Norris, 

 K.P. and Wilkins,M.H.F., Discussions 

 of the Faraday Society, 1950, No. 9, 

 360-363, Barer, R., Discussions of the 

 Faraday Society, 1950, No. 9, 369- 

 378, Mellors, R. C., Discussions of the 

 Faraday Society, 1950, No. 9, 398-406, 

 Thorell, B., Discussions of the Faraday 

 Society, 1950, No. 9, 432-436, Walker, 

 P. M. B. and Davies, H. G., Discus- 

 sions of the Faraday Society, 1950, 

 No. 9, 461-470, Commoner, B., Ann. 

 Mo. Bot. Card., 1948, 35, 239-254, 

 Burch, C. R., Proc. Physic. Soc, 1947, 

 59, 41-46, Grey, D. S., J. Opt. Soc. 



