Ultrathin (1 nm) vertically shadowed platinum‐carbon replicas for imaging individual molecules in freeze‐etched biological DNA and material science metal and plastic specimens

Abstract

Single molecule resolution in beam-sensitive, uncoated, noncrystalline materials has heretofore not been possible except in thin (less than or equal to 150 A) platinum-carbon (Pt-C) replicas, which are resistant to electron beam destruction. Previously, the granularity of metal film replicas limited their resolution to greater than or equal to 20 A. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low-angle 20 degrees rotary, 45 degrees unidirectional, and vertical 9.7 +/- 1 A Pt-C films deposited on mica under the same conditions were compared. Vertical replication had a 5 A granularity, the highest resolution, and evenly coated the whole surface. A 45 degrees replication had a 9.5 A granularity, a slightly poorer resolution, and a discontinuous surface coating. The use of 20 degrees rotary replication proved to be unsuitable for high-resolution imaging, with 20-25 A granularity and resolution two to three times poorer. Vertical and 45 degrees Pt-C replicas can visualize the deep-etched DNA helix and the 13.3 A 3(2) helix of pectin in a gel. The DNA double helix, the complex structures of sol-gel glasses, Immobilon filters (polyvinylidene fluoride), a polymethacrylate plastic, the metal oxide surfaces of 440c stainless steel, and aluminum are illustrated. This high-resolution vertical Pt-C replica technique can image in the context of solutions, gels, or solids, single molecular chains 3-7 A wide, their associations, and their conformation. Included in the present article are first time descriptions for removing replicas from metals and plastics and for making high-magnification photographic prints of normal contrast using a reversal rephotographic process.

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1

“…4]) and from the inherent loss of vertical surface structure which is caused by the multiple tunneling points that can occur when scanning over a high, angular structures (height = -15 nm; width = -10 nm) relative to the graphite substrate. Coatings (e.g., C or Pt) also enlarge the projection of the specimen from the substrate surface (such as hoops deposited on graphite) and fill in pores or depressions, thereby reducing their size (24).…”

Alzheimer disease hyperphosphorylated tau aggregates hydrophobically

“…The negatively stained images were printed directly from the TEM negatives. The TEM negatives from the replicas were used to make reversal negatives as described before (Ruben, 1989). The reversal negatives were printed on 8Љ ϫ 10Љ Ilford Multigrade II fiber based paper enlarged 2.5 times on a Durst 1200 point source enlarger with an Circular dichroism spectrum of ϳ160 µg/ml of AD P-tau #1 in 50 mM sodium phosphate, pH 6.5, as a function of temperature from 5-85°C.…”

“…The filter was removed from the centrifuge and washed thoroughly with distilled water in a Boyden chamber filter unit (Ruben et al, 1992). The filter was plunge-frozen in liquid propane cooled with liquid nitrogen and later freeze-dried at Ϫ80°C for 1.5 h in a modified Balzers 300, replicated with 0.93-1.0 nm Pt/C and coated with 12.1-12.8 nm rotary deposited carbon applied in two steps (Ruben, 1989). The replica was removed from the filter by floating the silver filter on a saturated potassium cyanide solution overnight or until the silver was dissolved.…”

“…The replica was floated on a saturated KOH solution overnight to remove the sample and then washed on distilled water for 24 h before being picked up on bare 300 mesh copper grids. A silver filter without any sample was also replicated in the Balzers 300 with 0.93 nm Pt-C and rotary carbon coated in two steps with 11.6 nm (Ruben, 1989). The silver filter was digested from beneath the replica as described and mounted on a bare 300 mesh copper grid.…”

“…Samples were either negatively stained on thin carbon films or vertically replicated (Ruben, 1989) on 0.2 µm silver filters. The negative staining technique is excellent for identifying large tau aggregates but is unreliable in imaging small aggregates of a few tau monomers.…”