QUANTIFICATION OF PARTICLE SIZES WITH METAL REPLICATION UNDER STANDARD FREEZE-ETCHING CONDITIONS - A GOLD BALL STANDARD FOR CALIBRATING SHADOW WIDTHS WAS USED TO MEASURE FREEZE-ETCHED GLOBULAR-PROTEINS

The real size of platinum-carbon (Pt-C) replicated particles is not di rectly equivalent to either its metal-coated diameter or its shadow wi dth. This paper describes two indirect methods, shadow widths and coat ed particle diameters, for determining a particle's actual size beneat h a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45 degrees angle on an similar to-100 degrees C surface in a 10(-6) torr vacuum. For the first method, gold balls nucleated in a partial pressu re of helium and deposited on flat indirect carbon films (root mean sq uare roughness of 0.8 nm) on 400 mesh grids were used as test particle s for calibrating shadow widths as a function of particle size. The go ld ball test specimens were replicated, and a distribution of Pt-C sha dow widths orthogonal to the Pt-C deposition direction was measured an d averaged for gold balls 1.5 +/- 0.25 nm, 2.0 +/- 0.25 nm, etc. The d iameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to t he gold. The shadow width distributions for each gold size do not diff er significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F-1, were also used as replication test objects. Negative staining of both proteins was conducted to measure t heir average diameters. In the second method, a distribution of Pt-C-c oated lac repressor diameters perpendicular to the shadow direction wa s measured. The Pt-C film thickness measured on the quartz crystal mon itor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. Whil e doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high con trast, granular Pt-C film, whereas 120 sec yielded a low contrast, les s granular Pt-C film. Both gold balls and protein particles were subje cted in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effe ct on the average size of the Pt-C shadow widths on both gold and prot ein particles. The Pt-C gun outgassing procedure also determined the s mallest replicated particle that could be resolved. The frequency of s ome smaller gold ball sizes detected after replication was reduced dis proportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-co ated protein particles. The ''geometric assumption'' that each metal-c oated particle creates a shadow width the same size as the metal-coate d particle diameter was tested using a globular protein. Pt-C replicat ion of protein particles at a 45 degrees and 20 degrees angle could no t confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle dia meter. A model for how the large shadow widths are formed is presented . Gold balls were also replicated at a 45 degrees angle with current h igh resolution conditions at a substrate temperature of -185 degrees C , and the results of these replicas were compared to the results repor ted here at similar to-100 degrees C. (C) 1995 Wiley-Liss, Inc.