Atomic Force Microscopy of Biomaterials, Mica, and the Origins of Life

Introduction Atomic force microscopy (AFM) began as an art as well as a science, and in many ways it continues to be an art and a science. Despite the existence of new, better, faster, bigger (or smaller!) atomic force microscopes (AFMs), some of the of the fundamental challenges remain—probe tips, sample dirt, and moving beyond leading-edge results obtained with one biological system to obtain leading-edge results with a related system. With regard to sample preparation, some methodological approaches are easily communicated. Other approaches are often nearly subconscious, such as the choice of rinsing a sample with a stream of water directed directly at the sample spot or directed above the sample spot, such that the water flows more gently over the sample spot. This can result in very different surface coverages, such that one person’s samples will have many DNA molecules per square micron, whereas another person’s samples will have only an occasional DNA molecule. Different regions of the same sample can appear very different. In one early experiment, our samples of DNA with a single-stranded binding protein showed only a solid “lawn” of material. By imaging progressively farther from the center of the sample, we eventually came upon a region where the lawn ended and individual molecules of DNA coated with singlestranded binding protein were seen. Operation of AFMs is also a combination of art and science that involves learning general rules for cantilever tuning, imaging forces, speeds, and gains and then manipulating these variables to optimize the imaging of the specific sample under the cantilever’s probe tip. Exciting breakthroughs were made in some of our earliest work, such as the time-lapse movie of fibrin polymerization [1] and reproducible imaging of DNA under propanol (Figure 1A) [2]. Later research included time-lapse movies of DNA (Figures 1B & 2) [3, 4], other research on DNA (Figure 1C) [5–8], the extracellular matrix [9, 10], synaptic vesicles [11, 12], analyses of bacterial biofilms [13, 14], and pulling on spider silk [15, 16]. AFM methods for imaging in fluid are presented in Kindt et al.’s article in Methods in Cell Biology [17], which is part of a useful volume about AFM methods in cell biology [18]. Several reviews by members of our lab have appeared therein [19–21]. Results Fly eye. An undergraduate in the lab caught a fly, and Oroudjev removed the eye, mounted it, and imaged it by AFM. Individual ommatidia in the compound eye of the fly are ~20 μm wide and ~3 μm high (Figure 3). The surfaces of the ommatidia are covered with aperiodic arrays of substructures, as seen in amplitude and phase images from tapping-mode AFM (Figure 4). These substructures have been described as a cerebral cortexlike pattern of ridges in earlier AFM images [22]. Collagenous extracellular matrix (ECM) from osteoblasts. Osteoblasts are bone-building cells that produce large quantities of extracellular collagen. Extracellular matrix from cultured osteoblasts was treated to remove calcium, as outlined in the caption of Figure 5. Collagen fibrils in the ECM can be identified by their strong banding, with 64to 65-nm periodicities and weaker banding with 32–33 nm. The 2 × 2 μm amplitude image inset in Figure 5B shows a region almost completely covered with nearly parallel collagen fibrils. Probe tips—still a holy grail of AFM research. Imagine that the AFM could reliably image sequence-dependent substructure on DNA at the level of the double-helix. This appears to have been done in a small series of images from our lab in the mid -1990s. Figure 6 is one of these images. This image was taken with an early prototype AFM for tapping in fluid