G. Mascetti et al., EFFECT OF FIXATIVES ON CALF THYMOCYTES CHROMATIN AS ANALYZED BY 3D HIGH-RESOLUTION FLUORESCENCE MICROSCOPY, Cytometry, 23(2), 1996, pp. 110-119
Two common fixatives-glutaraldehyde and ethanol/acetic acid mixture-we
re studied to understand their effects on DNA distribution inside the
cell nucleus. Native calf thymocytes were analyzed by using a DNA sele
ctive fluorescent dye (DAPI) and computational optical sectioning micr
oscopy on isolated cells before and after fixation, In order to estima
te quantitatively the intranuclear DNA distribution, the stained calf
thymocytes images were processed by removing the out-of-focus contribu
tions present in each optical section. Although preliminary, the resul
ts show that within individual nuclei the frequency distribution of th
e fluorescence intensity appears significantly and differentially alte
red by the two fixatives. Namely with respect to the native infixed pr
eparation, the ethanol/acetic acid causes the complete disappearance o
f the higher intensity pixels, whereas glutaraldehyde fixation can be
associated with the appearance of new ones of an even higher intensity
. The quantitative analysis of the processed images allowed us to reco
nstruct the topological distribution of DNA inside the nucleus and to
correlate the data with the results obtained by differential scanning
calorimetry on similar samples. (C) 1996 Wiley-Liss, Inc.