The distribution of the anti-cancer drug doxorubicin (DOX) in human breast cancer MCF-7 cells was imaged directly by low-energy-loss electron microscopy (EM) without specific antibodies or heavy metal stains, using only the electron-induced molecular orbital excitation of the drug. Cells treated with DOX were examined live by confocal fluorescence microscopy and as very thin sections in an electron microscope equipped with an electron energy filter having an energy resolution of 1 eV. The distribution of DOX obtained by EM from pairs of images at energy losses of 3 +/- 1 eV and 10 +/- 1 eV agreed with fluorescence microscope observations, but provided much more detail, easily distinguishing localization between nuclear membrane and perimembrane compartments and between vacuolated nucleoli and perinucleolar chromatin. Treatment times up to 1 h and DOX concentrations up to 30 mu M indicated a progression of DOX ingress from higher concentrations in the nuclear membrane to labeling of the nucleolus. Subsequently DOX moved into perinucleolar chromatin and concentrated in perimembrane chromatin aggregations. Quantification of the DOX signal indicated a decay half-life of 320 e/angstrom(2) under electron irradiation, whereas each image at 3000x required 10 e/angstrom(2). The results point to a new field of high resolution microanalysis: color electron microscopy. (c) 2006 Elsevier Inc. All rights reserved.