FLUORESCENCE STUDIES OF NUCLEOPROTEINS 125 



the ]m)toiii (partial de])roteinizati()n) and the initial stage of DNA 

 denaturation take ])lace witli transition of the DNx\ to a less rigid 

 stnictnre. This in tnrn facilitates the aggregation of the cations of 

 acridine orange bound with DNA {cf. Bradley and Felsenfeld, 11)59). 



Fluorescence microscopy in the visible region of the spectrum reveals 

 initial and progressive changes in the physico-chemical state of DNA, 

 in particular its separation from the chromatinic structures in the 

 irradiated cell nuclei and depolymerization. The changes may be de- 

 tected soon after irradiation in the hemopoietic organs and in the 

 leucocytes of the peripheral blood stream. 



2. By means of vital and supravital fluorochroming in hemojwietic 

 organs of irradiated animals one may observe peculiar focal cellular 

 degenerations — "micronecrotic foci". The nucleic acids of cells forming 

 such foci acquire enhanced affinity for diaminoacridines and brilliantly 

 fluoresce both in the visible (on fluorochroming) as well as in the ultra- 

 violet (primary fluorescence) regions of the spectrum. The physico- 

 chemical proi3erties of the nucleic acids and the nature of their 

 bonds with the cellular structures evidently differ significantly from 

 normal. 



3. In the cytoplasm of irradiated cells considerable accumulation of 

 nucleotides and nucleic acids takes place, some of them evidently 

 differing from the normal. These substances possess a clearly defined 

 ultraviolet autofluorescence, as a i-esult of which in irradiated cells that 

 continue their metabolic activities there is a marked intensification of 

 ultraviolet fluorescence. The fluorochrome acridine orange forms com- 

 plexes with nucleotides and RNA that separate out in the form of 

 granules with a red fluorescence. The content of such granules increases 

 to a great degi-ee in irradiated cells. They fluoresce (autofluorescence) 

 also in the ultraviolet region. Some of the granules in irradiated cells 

 are characterized by increased stability. On the other hand the majority 

 of the cytoplasmic nucleoproteins of irradiated cells become very labile 

 and sensitive to the short wave ultraviolet spectrum. 



4. Ultraviolet autofluorescence of animal cells and organs and of 

 blood plasma quickly diminishes under the influence of intensive ultra- 

 violet (A = 250 to 280 m/ii) and X-ray irradiation owmg to the occur- 

 rence of photochemical and X-ray processes. In parallel this fluor- 

 escence appears in the longer wave-length, visible part of the spectrum. 

 The appearance of this fluorescence is a sign of major changes in the 

 structure and state of the nucleoproteins and proteins. 



5. Ultraviolet fluorescence microscopy (Brumberg, 1956) first applied 

 to the study of radiation damage to cells yields particularly in com- 

 bination with ultraviolet absorption microscopy and fluorescence 



