Vertical Pt-C replication for TEM, a revolution in imaging non-periodic macromolecules, biological gels and...

Vertical replication for TEM is ideal for studying non-periodic specimens from 0.7 to 3 nm, a resolution mid-range difficult to attain by any other technique. This paper discusses the importance of vertical replication, its methods and hardware for high-resolution TEM. Evidence from diverse published research will demonstrate vertical replication's versatility in imaging the molecular level normally unattainable in freeze-dried polymers, polyethylene tribological wear on surfaces, low-density polymer networks or biological gels. Vertical platinum-carbon (Pt-C) replication minimizes the horizontal movement of Pt-C on a surface. Surface objects are symmetrically enlarged by a vertically deposited Pt-C film. To estimate real size in replicas, 16-25 particles or filaments need to be measured in calibrated transmission electron microscopy (TEM) images and reduced by a value less than the Pt-C film thickness measured with a quartz monitor. Continuous, vertically deposited Pt-C films are formed on mica at a deposition thickness of around 1.0 nm and on silver at a thickness of 0.4-0.5 nm. The distance between helical turns in poly(1-tetradecene sulfone) of 0.7 nm is the highest resolution achieved with vertical replication. Two polysulfones freeze-dried and vertically replicated on mica contained structures are predicted by indirect physical chemical methods to be present in solution. Polymer chains are fully Pt-C coated, with no uncoated gaps along chains. Some side-chains on the extended non-helical poly(1-tetradecene sulfone) are also detected. To estimate the real chain width, polymer chains measured in images are reduced by the Pt-C film thickness minus 0.5 nm. The polymer chain widths estimated from molecular models are in the same range of widths as those measured using the image size correction method. Also, it is possible to distinguish random coil proteins (chain width of around 0.5 nm) from an alpha-helix (chain diameter of about 1 nm) in vertically replicated samples on silver substrates. In the future, subnanometer resolutions below 0.7 nm should be possible. The resolution of vertical replication depends on the thickness of a continuous, amorphous Pt-C film. That thin, continuous 0.4-0.5 nm Pt-C films on silver substrates can be made suggests that a point-to-point resolution limit of around 0.28 nm in TEM may ultimately be approachable with replication.