X-RAY MICROSCOPY 



Practical Achievcmciils 



Micro ra(lio«iraphy of liiological Tis- 

 sues llijihiy Absorbing X-rays. It is 



natural to suppose that microradiography 

 of calcium-containing tissues (bones, teeth 

 and various calcifications) would be the 

 main subject in which the selective absorp- 

 tion of x-rays could prove its validity for 

 medico-biological research, and yet there 

 have appeared until recently comparatively 

 few publications, probably because of the 

 difficulty in obtaining thin slides of undecal- 

 cified calcium-containing tissues for which 

 the grinding was commonly used. One can 

 get thin pieces of calcium-containing tissues 

 with this method. However, it is sometimes 

 impossible either to get ground sections 

 thinner than 50 m (especially those of can- 

 cellous bone and pathological calcifications) 

 or to obtain serial sections of calcareous 

 material. These drawbacks, together with 

 the complicated techniques of grinding, 

 appear to be the main obstacles to a wider 

 use of this method. The introduction of 

 plastic as an embedding medium and the 

 manufacture of bone microtomes with heavy 

 knives made of extra hard steel, allow the 

 cutting of undecalcified bone in serial sec- 

 tions 10 /i and less with minimal damage to 

 calcareous material (about techniciues of 

 cutting see below^ in Supplement). This cut- 

 ting together with microradiography of ob- 

 tained sections allow much more complete 

 qualitative and quantitative evaluation of 

 calcium distribution than was possible be- 

 fore. 



The first approach to the study of bone 

 problems was made by Amprino and Eng- 

 strom (121) who used microradiographs of 

 compact human bone as well as of bones from 

 experimental material. Ground sections 

 20-50 fj. thick were radiographed with x-rays 

 2.5 to 4.0 A long. From their microradio- 

 graphs these authors made at least two 

 important conclusions: (1) they queried the 

 validity of the Ebner-Gebhardt theory (114) 

 which states that the cement substance is 



the only bearer of calcium salts in bone, 

 because they think that fibers are also cal- 

 cified; (2) they indicated that calcium im- 

 poverishment of bone during atrophic proc- 

 ess goes on without, at least to some extent, 

 the simultaneous destruction of the organic 

 matrix. Contemporary findings, arrived at 

 with the improved techniciue of micro- 

 radiography, confirm data of this early work. 

 The next year Engstrom and Engfeldt (57) 

 demonstrated in microradiographs the dif- 

 ferent calcium content in neighboring la- 

 mellae of the osteon. However, their tech- 

 niques did not permit a thorough analysis of 

 this finding. 



The different mineralization of lamellae 

 was also found by Davies and Engstrom 

 (43) in 1954. Again microradiographs were 

 made of sections too thick to permit fur- 

 ther conclusions on the structure of lamellae. 

 Vincent, as mentioned above, combined mi- 

 croradiography with autoradiography (af- 

 ter administration of S^- to dogs) and his- 

 tology (after previous decalcification). He 

 confirmed the results of Amprino and Eng- 

 strom, Davies and Engstrom in part re- 

 ferring to different calcification of bone ele- 

 ments. 



Stain historadiography (Bohatirchuk, 

 1957) (30) opened new possibilities in mor- 

 phological bone studies. As was mentioned 

 above, two properties differentiate this 

 method from earlier ones : serial sectioning of 

 undecalcified bone along with serial micro- 

 radiography of the obtained sections, and 

 the parallel coloring of the same sections 

 with histological dyes. Accordingh^ the 

 morphology of calcium-containing and cal- 

 cium-free bone elements may be compared 

 in microradiographs and histological speci- 

 mens (the latter free of the artifacts caused 

 by decalcification and disembedding). 



Bohatirchuk (33) in 1959 published re- 

 sults of using this method in his studies on 

 bone morphology. He showed that the main 

 depot of calcium in fine-fibered bone is pres- 

 ent in fibers or fibrils, which circle around 



608 



