Fluorescence microscopy* 



The examination of fluorescing prepara- 

 tions differs fundamentally from the general 

 method of microscopy in which the light 

 transmitted or reflected by the preparation 

 is observed. 



In fluorescence microscopy the prepara- 

 tion becomes self-luminous while the radia- 

 tion exciting the luminosity does not con- 

 tribute to the image formation but is 

 eliminated by barrier filters. The fluorescing 

 part of the preparation appears bright, usu- 

 ally colored, against a dark background. For 

 excitation, light is used of shorter wave- 

 length than that emitted by the preparation. 

 Thus blue and green fluorescence can only 

 be excited by ultraviolet (UV), while yellow 

 and red fluorescence may also be excited by 

 intense blue-violet (BV). 



Since only a small part of the incident 

 radiant energy is converted into fluorescent 

 Ught, it is necessary in fluorescence micros- 

 copy to employ the most intense sources of 

 light available. These however, besides the 

 excitation radiations of short w^avelength, 

 also emit light of greater wavelength which 

 would completely flood the relatively weak 

 fluorescences. 



Therefore two kinds of filters are a part 

 of every fluorescence microscope: (1) ex- 

 citation filters, which transmit in the illumi- 

 nating beam only the excitation radiation of 

 the total radiation emitted by the light 

 source; (2) barrier filters, which bar the fur- 

 ther passage of excitation radiation in the 

 imaging beam. 



Just as the inherent color of a substance 

 is due to its transmission or reflection of the 

 nonabsorbed Ught falling upon it, so likewise 

 a primary fluorescence, where it occurs, is 

 mainly a function of the chemical constitu- 

 tion. On this basis, guided by the presence 

 or absence of fluorescence of definite quality, 



* From a Carl Zeiss brochure, by permission. 



conclusions can be drawn concerning basic 

 chemical composition (fluorescence analysis). 

 Besides the inherent color, affinity for certain 

 stains may be characteristic. The use of 

 stains in histology is a familiar application 

 of topochemical staining technique. Sim- 

 ilarly the method can also be extended to 

 fluorescence and the affinity of so-called 

 fluorchromes for specific substances. Fluor- 

 chromes do not necessarily have a pro- 

 nounced color — in fact some of them are 

 practically colorless. But they brightly 

 fluoresce in a characteristic color when ex- 

 posed to appropriate exciting radiation. 

 Among others, the following are suitable: 



basic fluorchromes 



berberine sulfate, auramine, euchrysin, 

 acridine orange, coriphosphin, rivanol, 

 trypaflavin 



neutral fluorchromes (lipophil) 

 rhodamine B 



acid fluorchromes 



fluorescein, thiazine red, sulforhoda- 

 mine, primuline 



Various tissues or cellular constituents 

 show a specific affinity for the fluorchromes. 

 A peculiar phenomenon, occurring more fre- 

 quently with fluorchromes than with or- 

 dinary stains, is the so-called metachroma- 

 sia. By this is meant that different parts of 

 a specimen "stain" differently with the same 

 fluorchrome, both quantitatively and qual- 

 itatively, dependent on their chemical con- 

 stitution. 



But the application is not restricted to 

 histology. Fluorchromes are specially suit- 

 able above all in physiology for tracing the 

 transportation and distribution of metabo- 

 lites (absorption, imbibition, secretion, ex- 

 cretion, transportation) and their storage. 

 In general these examinations are carried 

 out in reflected light, since usually the organs 

 cannot be transilluminated. 



Organs and organelles plainly show fluores- 



332 



