Effect of fixatives on calf thymocytes chromatin as analyzed by 3D high-resolution fluorescence microscopy

Two common fixatives--glutaraldehyde and ethanol/acetic acid mixture--were studied to understand their effects on DNA distribution inside the cell nucleus. Native calf thymocytes were analyzed by using a DNA selective fluorescent dye (DAPI) and computational optical sectioning microscopy on isolated cells before and after fixation. In order to estimate quantitatively the intranuclear DNA distribution, the stained calf thymocytes images were processed by removing the out-of-focus contributions present in each optical section. Although preliminary, the results show that within individual nuclei the frequency distribution of the fluorescence intensity appears significantly and differentially altered by the two fixatives. Namely with respect to the native unfixed preparation, the ethanol/acetic acid causes the complete disappearance of 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 reconstruct the topological distribution of DNA inside the nucleus and to correlate the data with the results obtained by differential scanning calorimetry on similar samples.