Dark field imaging of biological macromolecules with the scanning transmission electron microscope

Abstract

A scanning transmission electron microscope (STEM) equipped with a field emission gun was employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent on the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. To reduce deleterious effects of the substrates on the image quality, a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films was developed. These films are approximately 15 .ANG. thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained) were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, 2.5-4.4 lattice spacings in the ferritin core were directly observed (in focus). The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs and corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 .ANG.)-1 layer lines indicated the effective resolution attained on these particles.