Improvement in high resolution image quality of radiation-sensitive specimens achieved with reduced spot size of the electron beam.

High resolution images of thin paraffin crystals and of purple membrane (i.e., crystalline bacteriorhodopsin) have been recorded with illumination spots that are confined to a size of 1000 to 2000 A. Images recorded in this way show three to five times greater contrast than do images which are recorded with more conventional flood-beam (ca. 3 micron diameter) illumination. In addition, optical diffraction patterns of small areas of such images show much better preservation of contrast in all directions than is normally the case. Both effects substantiate the idea that beam-induced movement of radiation-sensitive organic specimens has been a major factor which has caused low dose images to be greatly inferior in their quality compared to electron diffraction patterns of the same type of specimens. In spite of the marked improvement that is obtained, we often observe an unexplained degree of contrast variation within the small illuminated area, demonstrating that even further improvement in the quality of low dose images should be possible.