Metal Coating Techniques for Contrast Enhancement of Biological Macromolecular Complexes In HRSEM, STEM and TEM Image Modes

Modern field emission SEMs fitted with in-lens or near-lens (W.D. = l-3mm.) stages and above-lens SE detectors are capable of imaging 1-5 nm features at high voltage (15-30kV) and 5-10 nm features at low voltage (l-5kV). Ultrathin fine-grain refractory metal coatings were shown to optimize higher-solution (HR) secondary electron imaging of light element specimens especially biological samples. Ambient temperature dried specimens and frozen-hydrated cryo-specimens coated with continuous 1nm-thick metal films and staged in-lens have revealed biologically significant features of molecular dimension in tandem HRSEM and scanning transmission (STEM) modes. Although positive and negative stains have proved useful for general contrast enhancement of TEM specimens these stains alter many of our new genetically synthesized block protein copolymers. Therefore we have turned to non-evaporated metal coatings for contrast enhancement of these specimens in the TEM.</jats:p

Morphological Characterization of an Elastin-Mimetic Triblock Copolymer Utilizing CRYO-HRSEM

The elastin-mimetic tri-block copolymer, C5, with the polypeptide repeat [((VPAVG)(IPAVG)3(VPAVG))m((VPGVG)2(VPGEG)(VPGVG)3)n((VPAVG)(IPAVG)3(VPAVG))m] was produced via protein hyper-expression utilizing a pET plasmid and Escherichia coli (BL21- Gold(DE3)) as the bacterial host. The gene coding for the polypeptide was produced by a multimerization protocol that has undergone recent modifications. A sample of the elastinmimetic block copolymer, C5, was prepared as a 20 to 25 percent protein solutions in sterile ddH20. Approximately 10 μL of the solution was pipetted into 3 mm gold planchets (Balzers BU 012 130T), which had been pre-equilibrated to 4°C in an isothermal environmental cooler (Torrey Pines Scientific). The temperature of the cooler was raised to 20°C and allowed to stabilize for 10 minutes. The solidified samples, were plunge-frozen in liquid ethane at its melting point (−183°C); the samples were stored in liquid nitrogen (LN2). A sample was transferred to and mounted on the precooled (∽−170°C) Oxford CT-3500 cryo-stage held in the cryo-preparation chamber.</jats:p