HISTOLOGY BY THE PROJECTION MICROSCOPE 



advantage is taken of the ultra-fine focus 

 and x-ray brilliance provided by the tube to 

 reduce geometrical unsharpness and expo- 

 sure time, while in the latter case full advan- 

 tage is taken of the resolution and primary 

 magnification afforded by the x-ray point 

 source. 



Histological sections can be examined with 

 the x-ray microscope under atmospheric 

 conditions by the projection method. Or- 

 dinary sections (10 -15 microns thick), 

 mounted on thin Terylene or Melinex plastic 

 film and deparaffinized with xylene and sev- 

 eral changes of ethyl alcohol, when placed 

 a short distance above the target (10 mm) 

 give very contrasty low power projection 

 micrographs (1 X to 20 X ) on standard lantern 

 slide emulsion (Ilford Contrasty plates). 

 Such micrographs show the varying radio- 

 densities of the different tissues and provide 

 much useful microanatomical information. 

 For example, transverse sections of human 

 foetal neck (C.R. 161 mm) when so exam- 

 ined show the lining epithelium of the laryn- 

 geal cleft, mural constituents of the oesopha- 

 gus, mature cartilage cells surrounding the 

 primary ossific center of the vertebral body, 

 as well as many other details of the cervical 

 musculature, vessels and nerves. Or again, 

 projection micrographs of the adult human 

 lung demonstrate the terminal pulmonary 

 anatomy almost diagrammatically, record- 

 ing the respiratory bronchioles, atria and 

 alveoli, as well as foreign inclusions such as 

 mine dusts (e.g., tin). 



However, tissue sections prepared by con- 

 ventional methods considered adequate for 

 optical microscopy are not suited to x-ray 

 microscopy which, like electron microscopy, 

 demands special attention to tissue prepara- 

 tion. Also absorption b}^ the plastic mounting 

 film and air column limit cellular definition, 

 and require the use of a vacuum camera. 



The vacuum camera used for high power 

 studies of tissue sections resembles a funnel 

 centered upon and threaded to the target 

 assembly of the x-ray microscope. The inte- 



rior of the camera is stepped in millimeter 

 stages to give known target to specimen 

 distances, and is fitted with specimen rings 

 for the support of tissue sections. The sec- 

 tions (4-6 microns thick) are mounted on 

 brass rings bearing a f ormvar film ; nonmag- 

 netic rings are essential since the section lies 

 within the magnetic field of the objective 

 lens polepiece. The formvar substrate is 

 best dispensed with in order to eliminate 

 extraneous absorption and sharpen cell defi- 

 nition. The upper part of the camera con- 

 sists of a removable photographic plate 

 chamber (4 cm high) that is evacuated by 

 a vacmmti pump. Ultra-fine grain emulsion, 

 such as maximum resolution plates (Kodak) 

 or Lippman film (Gevaert), should be used 

 for recording tissue micrographs. 



A certain primary magnification of 2 X to 

 10 X of unstained animal and human tissues 

 and cells can be obtained rapidly (,^^-4 min) 

 with the aid of such a camera, while operat- 

 ing the x-ray microscope at approximately 

 7 kv with a beam current of 4 to 100 micro- 

 amperes and using a thin aluminum target 

 (4 to 10 microns). Lower initial magnifica- 

 tion is used in the interests of intensity and 

 exposure time. Further useful secondary 

 magnification up to llOOX can be made on 

 fine grain emulsion (Kodak Microfile or 

 AdoxK.B. 14 or 17). 



In projection microscopy, resolution de- 

 pends upon the size of the x-ray source and 

 hence also partly upon focusing accurac3^ 

 Focusing is unfortunately critical and diffi- 

 cult at the lower kilovoltages so essential to 

 the production of reasonable contrast in 

 tissue sections. It can however, as mentioned 

 earlier, be facilitated by reducing the target- 

 screen distance before viewing the test grid. 



Other factors influencing sharpness of 

 definition and contrast, are the choice, fixa- 

 tion, and preparation of the tissue. Tissues 

 that undergo keratinization (skin) or min- 

 eralization (teeth, bone) give contrasty pro- 

 jection micrographs, as do those which 

 accumulate chemical elements sufficiently to 



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