Microscopy negative contrast

Double-helical DNA in solution can undergo strand separation or dena-turation as a consequence of extremes of pH, heat, or exposure to chemicals such as urea or amides. Decrease in viscosity, increase in absorbance at 260 nm (hyperchromic effect), decrease in buoyant density, or negative optical rotation indicates denaturation of DNA. The denaturation process disrupts only noncovalent interactions between the two strands of DNA. Since G-C base pairs are held together by three hydrogen bonds in contrast to two for an A-T base pair, A-T rich DNA is easily denatured compared to G-C rich DNA (Figure 6.3). Electron microscopy can detect these A-T-rich regions in a DNA molecule since they form bubblelike structures. Hence the temperature of melting (Tm) of DNA increases in a linear fashion with... 

For a high contrast in the electron micrographs, the sample was stained negative with uranyl acetate. This leads to denaturation of the enzyme, and this might be connected with considerable changes of shape and structure of the protein. In order to avoid these problems, cryo-electron microscopy of frozen-hydrated samples has been applied, similar to that carried out with FqFj from E.coli (6). 

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