GENEKAL MICROSCOPY 



In general, microspectrophotometry is ac- 

 complished in two ways. One is by illuminat- 

 ing the specimen with white light, and subse- 

 quently analyzing the light transmitted by 

 the subject. The other is by illuminating the 

 specimen with monochromatic light and 

 monitoring the sample transmittance as the 

 wavelength is varied. Jelley's microspectro- 

 graph (15) is an example of the first method. 

 He uses a transparent replica of a grating to 

 break down into its component wavelengths 

 the light transmitted by the specimen, and 

 records the spectra photographically. The 

 second method requires a monochromatic 

 light source, special optics if the range is to 

 be extended into the ultraviolet and infrared, 

 and suitable sensing devices for the wave- 

 lengths encountered. As usual, as the refine- 

 ments are made, the complexity of instru- 

 mentation becomes formidable. Some of the 

 Perkin-Elmer spectrophotometers are suited 

 to alteration for microspectrophotometry 

 through a commercially available micro- 

 scope accessory. Beckman also has provision 

 for a similar modification of its equipment. 

 It is anticipated that specialized equipment 

 of this type soon will be developed and made 

 available. 



Microspectrophotometry is especially use- 

 ful for the identification of organic com- 

 pounds or, more particularly, the chemical 

 groups found in these compounds. Spectra 

 in the infrared are more definitive ; neverthe- 

 less, significant comparisons can be made by 

 means of ultraviolet and visible spectra 

 among certain specimens giving information 

 as to the course of chemical reactions. 



Physical properties of small quantities of 

 materials can be determined microscopically. 

 Melting point, sublimation temperature, 

 hardness, solubility, deformation behavior, 

 and tensile strength are some of these. 



For example, the tensile strengths of single 

 textile fibers have been measured with the 

 aid of a microscope and a modified micro- 

 tensiometer. The torsion wire tensiometer, 

 conventionally used for surface tension 



measurements, was modified by the addition 

 of a lever arm. The extension of an attached 

 fiber could be observed directly in the mici'o- 

 scope. This apparatus was found (juite suit- 

 able for exerting force of the proper magni- 

 tude and with the desired control to make 

 tensile tests on individual wood pulp and 

 cotton fibers. The fibers were attached to the 

 tensiometer under a stereobinocular micro- 

 scope (IG). 



Melting points, sublimation temperatures, 

 and fusion data in general can be accumu- 

 lated with a heating or cooling stage. W. C. 

 McCrone (17) in his book "Fusion Methods 

 in Chemical Microscopy" has presented 

 many of the possibilities in this area. He 

 covers observations of temperature-depend- 

 ent phenomena of crystalline compounds 

 individually and in mixtures. Mixed fusion 

 of the unknown with a standard reference 

 compound has been emphasized. These 

 methods are suited to organic chemical 

 analysis. 



Fluorescence microscopy (19) involves 

 physicochemical phenomena from which 

 chemical inferences are drawn. Aside from 

 inherent fluorescence to be found in certain 

 specimens, fluorescence can be created by 

 suitable dyes which selectively attach to 

 specific compositions within a structure. 

 These dyes are called fluorochi^omes, and are 

 used by biologists, for example, in im- 

 munological chemistry, to locate structures 

 in organs which are most active in antibody 

 production. The fluorochromes are designed 

 to form true chemical bonds (not adsorption 

 complexes) with the antigenic protein sub- 

 stances which are collected by certain organs. 

 It is proposed that by this fluorochi-ome tech- 

 nique, selected nonbiological substances 

 could be traced thi-ough a chemical and 

 physical process. One of the outstanding 

 advantages of this method is the high sensi- 

 tivity. Dyestuffs in concentration as low as 

 jQ-is g pg^j^ \yQ detected. 



Microscopes for fluorescence observation 

 are supplied with ultraviolet illumination to 



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