Apoptotic cell nuclei are known to stain hyperchromatically with absor
ption dyes and dimly with many DNA fluorochromes. We hypothesised that
both optical phenomena have the same cause - the ability of apoptotic
chromatin to aggregate cationic dyes. This hypothesis was tested usin
g prednisolone-primed rat thymus, which is known to contain apoptotic
cells. The apoptotic cells were classified as early and late, based on
their morphology, in thin and semithin sections and in thymus imprint
s on slides. Direct reaction for DNA strand breaks (TUNEL) indicated t
he presence of breaks in both categories of cells, with more intense l
abelling in late apoptosis. The chromatin ultrastructure of early apop
totic cells initially retained the supranucleosomal order of packaging
which characterises control cells, whereas the dense chromatin of lat
e apoptotic cells possessed the degraded structure. Absorption spectra
of the toluidine blue-stained early apoptotic cell chromatin revealed
a metachromatic shift, indicating a change of DNA conformation and po
lymerisation of the dye. When the staining was performed by acridine o
range (preceded by a short acid treatment), a paradoxical several-fold
increase of fluorescence intensity at a several-fold dilution of the
dye was found. The simultaneous reduction of the ratio of red to green
components of fluorescence confirmed that the concentration-dependent
fluorescence quenching was due to aggregation of the dye. The results
suggest that the enhanced affinity of the chromatin of early apoptoti
c cells for cationic dyes is associated with conformational relaxation
rather than degradation of DNA. In late apoptotic cells, the very den
se packaging of degraded DNA promotes further aggregation of dyes. The
results suggest alternative methods for detection and discrimination
of early and late apoptotic cells.