Visualization of substructure in ferritin molecules: an artifact

It has been suggested in the literature that the core of the ferritin molecule contains a tetrad or other substructure resolvable in electron micrographs. The work described in this paper indicates : 1. At a potential resolution of 5 A, near-focus electron micrographs of ferritin molecules show no substructure in the core. 2. By defocusing the microscope a computed amount, electron micrographs of the same ferritin molecule can be produced which show apparent core substructure similar to that shown in several patterns reported in the literature. 3. Apparent ferritin molecules with or without substructure, depending upon the phase granularity in the negative, can be added in printing by over-exposing an area of the background equal in size to a ferritin molecule. 4. Underfocus contrast enhancement, frequently used by electron microscopists at lower magnifications without loss of resolution, does result in a loss of resolution at maximum useful magnifications. 5. The established physical principles of electron-optical image formation which should be applied to all high-resolution electron microscopy to distinguish substructure from artifact, are reviewed. This work is compatible with recent X-ray diffraction studies of ferritin molecules which indicate that iron-containing micelles in the core of ferritin are smaller than the practical resolution of the best electron microscopes and therefore neither they nor their substructure could be imaged.