High-resolution scanning tunneling microscopy of fully hydrated ripple-phase bilayers

A modified freeze-fracture replication technique for use with the scanning tunneling microscope (STM) has provided a quantitative, high-resolution description of the waveform and amplitude of rippled bilayers in the P beta' phase of dimyristoylphosphatidylcholine (DMPC) in excess water. The ripples are uniaxial and asymmetrical, with a temperature-dependent amplitude of 2.4 nm near the chain melting temperature that decreases to zero at the chain crystallization temperature. The wavelength of 11 nm does not change with temperature. The observed ripple shape and the temperature-induced structural changes are not predicted by any current theory. Calibration and reproducibility of the STM/replica technique were tested with replicas of well-characterized bilayers of cadmium arachidate on mica that provide regular 5.5-nm steps. STM images were analyzed using a cross-correlation averaging program to eliminate the effects of noise and the finite size and shapes of the metal grains that make up the replica. The correlation averaging allowed us to develop a composite ripple profile averaged over hundreds of individual ripples measured on different samples with different STM tips. The STM/replica technique avoids many of the previous artifacts of biological STM imaging and can be used to examine a variety of periodic hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm. This resolution is superior to conventional and tapping mode AFM to soft biological materials; the technique is substrate-free, and the conductive and chemically uniform replicas make image interpretation simple and direct.