| We have designed, constructed, and implemented an unusual Scanning Force Microscope (SFM), which we hope may address some of the difficulties encountered in the imaging of biological materials. The SFM (also known as the Atomic Force Microscope) is capable of extraordinary resolution, but has been limited in biological investigations by a lack of sample stability and reproducibility. Our SFM operates at temperatures as low as 143 K in a liquid pentane bath, which may increase both sample rigidity and stability, minimizing the problems encountered by other researchers, and opening the door for techniques such as freeze-fracture. We have successfully applied our low temperature SFM (LT-SFM) to a wide variety of biological materials including purple membrane, ferritin, and DNA. Our primary biological focus was on type I collagen, an important and ubiquitous protein. Collagen was selected given to its unusual and advantageous physical characteristics, previous experience including Scanning Tunneling Microscopy (STM), and its intrinsically interesting nature. Our efforts represent some of the highest resolution images currently available for native macromolecular structures, and our collagen monomer micrographs appear to be the first obtained with SFM (Shattuck et al., 1992), both at room temperature and at 143 K. We are aware of several attempts at imaging collagen monomers with the SFM, most notably the work of Chernoff & Chernoff (1992), but apparently to date these studies have only resolved larger fibrillar structures. Indeed, molecular biological SFM observations have been quite limited. Similarly, a previous LT-SFM, constructed by Prader (1991) in Hansma's group, was not successful in obtaining images of single molecules. Our collagen data is of sufficient quantity and quality that measurements of several intermolecular parameters and features are possible, with correlation to the known physical and biochemical properties of the molecule (Shattuck et al., 1994), including charged molecular domains. We verified our collagen work with transmission electron microscopy and gel electrophoresis. Additionally we were able to extend our studies to direct physical manipulation at the molecular level, including machining of biological structures, measurements of molecular elasticity, and preliminary freeze-fracture studies. |
