ROENTGEN ABSORPTION HISTOSPECTROSCOPY 127 



D. ROENTGEN ABSORPTION 

 HISTOSPECTROSCOPY* 



One of the most significant advances in histo- and cytochemical 

 technique has come from the work of Engstrom ( 1946) at Karohnska 

 Institutet, Stockhohn, who, by employing the roentgen absorption 

 of tissue sections or of very small volumes of liquid, developed a 

 procedure whereby quantitative elementary analyses can be directly 

 ]ierformed with an accuracy of about 5-10% on 1 X 10"^ to 1 X 

 10"^- gram of material, i.e., quantities of the order found in single 

 mammalian cells. Thus, in specific instances, phosphorus and calcium 

 can be determined in a 10 /x section of bony tissue within an area 

 around 10 X 10 ix, and nitrogen and oxygen in a section a couple 

 of microns thick within an area of 50-100 /x-. A particular advantage 

 of this technique is that the tissue is not used up, and hence may be 

 subsequently employed for histological study by the usual methods 

 so that direct correlation may be made between the chemical 

 composition and the microanatomical structure. Furthermore, the 

 analysis is independent of the chemical structure in which the ele- 

 ment may be bound, and the physical state of the specimen is 

 unimportant, e.g., fixed tissue, dry powder, and in certain cases, 

 paraffin-embedded tissue, or solutions, may be used. A number of 

 elements can be determined on the same sample. 



The advantages of being able to determine the total quantity of 

 a tissue element in situ without regard to its chemical form and state 

 of valence, or affiliations with other elements, are hardly to be 

 minimized. The method is confined to the quantitative determination 

 of elements having an atomic number of 6 (carbon) or greater. This 

 would include all elements of biological importance with the 

 exception of hydrogen. 



Engstrom (1946) has pointed out that roentgen spectroscopic 

 methods based on emission analysis are not well suited for elements 

 with atomic numbers less than 20, and the emission methods cannot 

 be applied very well to the small surfaces involved in histo- and 

 cytochemical studies. This applies to the earlier procedure of von 

 Hamos and Engstrom ( 1944) in which tissue is subjected to roentgen 

 radiation and the secondary radiation is measured for the quantita- 

 tion of constituent elements. 



* See Bibliography Appendix, Refs. 23, 24, 25, 26, and 27. 



