Protein Visualization with Crystal Contrast

Good contrast can be obtained for unstained protein in biological molecules when the molecules are supported on top of a single crystal film oriented so as to strongly diffract the illumination electrons. If the objective aperture excludes the diffracted beam, the strongly diffracting regions in the crystal film will show up in the image as dark bands a micron or more wide known as bend contours. The bend contour width is determined by the wrinkling of the crystal film which causes local changes in the crystal support film orientation. Biological molecules viewed near bend contours show contrast at exact focus without requiring staining or shadowing with heavy metal atoms. The bend contours are easily moved around by tilting the sample with a tilt stage so that it is not difficult to orient the crystal support so that it is strongly diffracting where ever the biological molecules are located on the support film. The details of the contrast mechanism have been discussed by Hines and Howie.