(a) GENERAL INFORMATION 



Fluorescence is the property possessed by many substances of 

 converting short wavelengths of Hght into longer wavelengths. In 

 microscopy the substances and structures of most interest are 

 those which convert ultraviolet light into light of the visible spec- 

 trum, as only substances of this character can be observed directly. 

 It is, of course, well known that most living organisms are pro- 

 foundly affected by short light waves, and a great deal of infor- 

 mation as to their structure has been obtained by the study 

 of the appearance of these organisms under the influence of 

 invisible light rays. If individual cells or structural units are 

 examined before, during, and after ultraviolet treatment, enough 

 of this effect should be discovered to impart some under- 

 standing as to the changes which occur in the animal, or plant, 

 as a whole. 



In fluorescence microscopy structural details are rendered 

 visible by: {a) innate auto-fluorescence, a property possessed by 

 most tissues which when excited by short light waves become 

 clearly visible since they become luminous and glow or " fluor- 

 esce " with a radiance of their own, or by {h) secondary fluorescence 

 which is known as " fluorochromy " and is brought about by the 

 process of treatment of the tissues with fluorescent dyes and cer- 

 tain alkaloids (e.g. Berberine sulphate) and other substances. 



It is proposed to deal only with fluorochromy in the short space 

 of this chapter. 



Fluorescent dyes and other substances used for this purpose are 

 known collectively as " fluorochromes " ; these materials are 

 selectively absorbed by certain parts of the cell. When tissues, 

 bacteria or protozoa, which have undergone treatment with fluoro- 

 chromes are examined under the microscope, using ultraviolet 

 light instead of transmitted light of the visible spectrum, they 

 become visible as bright luminous objects against a dark back- 

 ground. Cells stained with fluorochromes absorb the ultraviolet 

 rays of short wavelength, and emit this energy in the form of 



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