HIGH-RESOLUTION SCANNING-TUNNELING-MICROSCOPY OF FULLY HYDRATED RIPPLE-PHASE BILAYERS

A modified freeze-fracture replication technique for use with the scan ning tunneling microscope (STM) has provided a quantitative, high-reso lution description of the waveform and amplitude of rippled bilayers i n 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 wavele ngth of 11 nm does not change with temperature. The observed ripple sh ape and the temperature-induced structural changes are not predicted b y any current theory. Calibration and reproducibility of the STM/repli ca technique were tested with replicas of well-characterized bilayers of cadmium arachidate on mica that provide regular 5.5-nm steps. STM i mages were analyzed using a cross-correlation averaging program to eli minate the effects of noise and the finite size and shapes of the meta l grains that make up the replica. The correlation averaging allowed u s to develop a composite ripple profile averaged over hundreds of indi vidual ripples measured on different samples with different STM tips. The STM/replica technique avoids many of the previous artifacts of bio logical STM imaging and can be used to examine a variety of periodic h ydrated lipid and protein samples at a lateral resolution of about 1 n m and a vertical resolution of about 0.3 nm. This resolution is superi or to conventional and tapping mode AFM of soft biological materials; the technique is substrate-free, and the conductive and chemically uni form replicas make image interpretation simple and direct.