104 MICROSCOPIC TECHNIQUES 



4. Characterization of Substances 



Direct Observation of Fluorescence 



Vitamin A. Particularly intensive work has been done on vita- 

 min A, starting with the work of von Querner (1935), Hirt and 

 Wimmer (1940), and others, and continuing in greatly expanded 

 scope and development with the research of Popper (1944) and 

 associates. The fading green fluorescence of vitamin A in tissue 

 sections has been found to run parallel with the results of chemical 

 determination (Popper and Elsasser, 1941). The fading green fluo- 

 rescence is characteristic of vitamin A], found in salt water fish, and 

 a slowly fading pale yellow-brown fluorescence characterizes vitamin 

 A2, found in fresh-water fish. An admirable review of the studies 

 made on vitamin A distribution in the tissues of animals and man, 

 both in normal and pathological states, has been presented by Popper 

 (1944). He pointed out that carotene may easily be differentiated 

 from the A vitamins by its very slowly fading green fluorescence 

 which is apparent only in higher concentrations, and that the bio- 

 logically inactive anhydro ("cyclized") vitamin A may be recog- 

 nized by its dark brown fluorescence which gradually becomes a dull 

 green and finally fades out entirely. Volk and Popper (1944a) re- 

 ported the existence of a factor in biological fluids, particularly 

 plasma, and in organ emulsions that delays the disappearance of 

 vitamin A fluorescence in tissue sections. 



Riboflavin. EUinger and Koschara (1933), von Euler et al. 

 ( 1935) , Hirt and Wimmer ( 1939a) , and Metcalf and Patton ( 1942) 

 utilized the yellowish-green fluorescence of riboflavin for its identi- 

 fication in tissues. According to Ellinger (1938), and confirmed by 

 Metcalf and Patton (1942), another form of riboflavin exists (prob- 

 ably bound to another compound) which gives a yellow-orange 

 fluorescence. INIetcalf (1943> subsequently concluded that in the 

 American roach, Periplaneta americana L., the bound riboflavin is 

 converted to the free form in vivo by the injection of pantothenic 

 acid or thiamine. With the latter compound the conversion proceeds 

 more slowly. 



Other Vitamins.* Attempts have been made to characterize, and 

 to determine the distribution of, other vitamins by their fluorescent 

 properties. Hirt and Wimmer ( 1939b) investigated nicotinic acid 

 and its amide which they claimed gave a stable yellow fluorescence. 



* See Bibliography Appendix, Ref . 29. 



