X-RAY MICROSCOPY 



As discussed by Pattee in his article on 

 Microfluoroscopy, almost grainless thin 

 films of fluorescent materials can be pro- 

 duced, thus providing excellent resolution 

 at high magnifications. 



An important improvement in the polymer 

 film method was announced in the July 25, 

 1960 number of Chemical and Engineering 

 News. This was achieved by Dr. Saara 

 Asunmaa, working under Dr. H. H. Pattee, 

 Jr. in Stanford University's Biophysics Lab- 

 oratory. 



Dr. Asunmaa makes a contact x-ray mi- 

 crograph on a sensitive film in which the 

 absorbed radiation changes the electron 

 density. This film is then used as a specimen 

 in the electron microscope. The film that 

 does the trick is made of cellulose nitrate 

 activated with silver chloride. Dr. Asunmaa 

 mixes lithium chloride and silver nitrate in 

 an acidic medium with a 2% solution of 

 cellulose nitrate in ethanol and ether. From 

 this she casts the thin films^ — about 1000 A. 

 thick — on glass slides, then ages them in a 

 desiccator. 



To make the micrograph she places the 

 specimen in contact with the film and ex- 

 poses it to x-rays from a microfocus tube for 

 10 minutes to one hour. The silver chloride 

 particles act as radiation traps and are re- 

 duced to metallic silver. The reduced silver 

 gives an image that is directly visible imder 

 a light microscope, but this optical contrast 

 does not correspond to differences in electron 

 scattering. 



To get an image that is observable in the 

 electron microscope, the structure of the film 

 must be altered. The radiation absorbed by 

 the silver chloride not only reduces the silver 

 salt but also degrades the cellulose nitrate. 

 Aqueous methanolic sodium cyanide dis- 

 solves the soluble low molecular weight 

 fraction of the polymer, while the silver and 

 unreduced silver salts are removed as cy- 

 anide complexes. 



What is left is a relief image with electron 

 optical contrast corresponding to x-ray ab- 



sorption by the original specimen. This reUef 

 image can now be used as the specimen in an 

 electron microscope. The enlargement here 

 is limited only by the resolution obtained in 

 the original x-ray micrograph. Using diatoms 

 as test specimens Dr. Asunmaa has demon- 

 strated resolution down to at least 600 A. 

 and has produced electron micrographs with 

 enlargements up to 40,000 diameters. 



REFERENCE 



1. Ladd, W. a., Hess, W. M., and Ladd, W. M., 

 Science, 123, 370 (1956). 



G. L. Clark 



HISTOLOGY BY THE PROJECTION 

 MICROSCOPE 



Historadiography (1) or the study of tis- 

 sues and cells by x-rays, has to date been 

 carried out almost exclusively by contact 

 microradiography. Using preparation tech- 

 niques generally identical with those used in 

 optical microscopy, the thin tissue section 

 is either placed upon or embedded in the 

 emulsion of a fine grained photographic 

 plate and exposed to soft x-radiation. The 

 small x-ray picture or microradiogram so 

 obtained at unit magnification is subse- 

 quently magnified optically. 



A major problem (2) has been the attain- 

 ment of adequate resolution, since ordinary 

 cells measure only about 10 microns in diam- 

 eter, and structures within the cells are less 

 than 5 microns in diameter. So far as mi- 

 croscopy is concerned the contact method 

 is limited by the grain size of the recording 

 emulsion, size of the x-ray source, and resolu- 

 tion of the optical system used for viewing 

 or enlarging the plate image. 



The x-ray projection microscope has both 

 theoretically and actually a high resolving 

 power, and a resolution of 0.05 micron seems 

 practicable in the near future. Tissue sec- 

 tions can be studied with the instrument 

 using either the contact or projection 

 method. In practicing the former method. 



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