Most cells treated with 5 μM sanguinarine for 24 hours are not apoptotic. Multiparameter imaging was conducted with control (a-d) and sanguinarine-treated (e-h) cultures, using differential interference (DIC) microscopy (a and e), a fluorescein filter set to visualize calcein (green, b and f), a rhodamine filter set to detect both TMRM and ethidium homodimer (red, c and g), and a UV filter set to show Hoechst (blue, d and h). The same field of view is shown for each of the imaging methods for the control and drug-treated samples. Living cells fluoresce green and contain red mitochondria, but lack red nuclear labeling. In contrast, apoptotic cells do not display calcein or mitochondrial labeling, but contain nuclei with condensing chromatin that are both red (indicating ethidium homodimer penetration) and blue. Two apoptotic cells are present in the sanguinarine sample (arrows). The nucleus of the lower apoptotic cell consisted of a tightly condensed ball of heterochromatin, which is mostly out of the plane of focus in this image. Apoptotic cells can be found in both control and sanguinarine cultures, but are easily identifiable, even solely by DIC microscopy. Only healthy appearing, well-spread cells were used to show the redistribution of cyclin D1 and topoisomerase II in sanguinarine-treated cultures (Figs. 2 and 3).