Woodward J T, Zasadzinski J A
Department of Physics, University of California, Santa Barbara 93106-5080, USA.
Scanning Microsc Suppl. 1996;10:123-47; discussion 147-8.
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.
通过改进冷冻断裂复型技术（一种标准的电子显微镜固定技术），使其适用于扫描隧道显微镜（STM），可以对各种柔软的非导电生物材料进行三维高分辨率成像。与原始生物材料相比，金属复型是STM近乎理想的样品。改进措施包括在银上制备0.1微米厚的背衬层，并将复型安装在细网银滤器上，以增强金属复型的刚性。除非在真空中进行STM成像，否则这是必需的；否则，污染液膜会使STM探针与样品物理连接。这种机械耦合会导致高度测量值夸大；较厚复型增强的刚性消除了大部分高度放大效应。在干燥氮气气氛中成像可进一步改善效果。使用在云母上具有特征明确的花生酸镉双层复型进行校准和再现性测试，该双层提供规则的5.5纳米台阶。我们已使用STM/复型技术来研究水中二肉豆蔻酰磷脂酰胆碱（DMPC）的Pβ相中的波纹形状和幅度。使用互相关平均程序分析STM图像，以消除噪声以及构成复型的金属颗粒的有限尺寸和形状的影响。相关平均使我们能够生成一个在数百个单独波纹和不同样品上平均的复合波纹轮廓。STM/复型技术具有足够的通用性，可用于以约1纳米的横向分辨率和约0.3纳米的垂直分辨率检查各种水合脂质和蛋白质样品。
