Many issues in the examination, treatment, and authentication of works of art depend on the accurate characterization of thin layers, which may challenge the resolution and detection limits of instrumentation routinely used for analyses, particularly SEM-EDS and EPMA. Such thin layers are the focus of recent conservation analysis in preparation for a major retrospective of Thomas Eakins’ works. Interpretation of Eakins’ paintings has often been complicated by mechanical and chemical cleaning procedures performed after the artist’s death in 1916. Recent advances in Focused Ion Beam (FEB) technology provide means for preparing specimens of thin layers that can be analyzed directly by transmission electron microscopy (TEM) yielding resolution in the nanometer range or better. This paper describes initial work undertaken to elucidate the inorganic components in the uppermost paint layers from Eakins’ The Crucifixion, completed in 1880. Using TEM, we have observed a 250-500 nm Pb-rich nanocrystalline region, a pigment free zone less than 2 ?m thick, and a lead white paint layer. An analysis of samples from two paintings that were subjected to mild cleaning operations did not show comparable Pb-rich nanocrystals. These results suggest that The Crucifixion may have been subjected to a more aggressive cleaning treatment which caused the entrainment of the Pb-rich material – not resolvable by traditional analytical techniques – at the painting’s surface. The use of FIB/TEM has enabled clear identification of the thin paint surface layers and offers enormous promise for understanding the processing and alteration of artists’ materials, including issues of authentication.