Imaging soft materials with scanning tunneling microscopy

By modifying freeze-fracture replication, a standard electron microscopy fixation technique, for use with the scanning tunneling microscope (STM), a variety of soft, non-conductive biomaterials can be imaged at high resolution in three dimensions. Metal replicas make near ideal samples for STM in comparison to the original biological materials. Modifications include a 0.1 micron backing layer of silver and mounting the replicas on a fine-mesh silver filters to enhance the rigidity of the metal replica. This is required unless STM imaging is carried out in vacuum; otherwise, a liquid film of contamination physically connects the STM tip with the sample. This mechanical coupling leads to exaggerated height measurements; the enhanced rigidity of the thicker replica eliminates much of the height amplification. Further improvement was obtained by imaging in a dry nitrogen atmosphere. Calibration and reproducibility were tested with replicas of well characterized bilayers of cadmium arachidate on mica that provide regular 5.5 nm steps. We have used the STM/replica technique to examine the ripple shape and amplitude in the P beta phase of dimyristoylphosphatidyl-choline (DMPC) in water. 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 and different samples. The STM/replica technique is sufficiently general that it can be used to examine a variety of hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm.