Scanning transmission electron microscopy
Stock β2m fibril solutions were either diluted 20× to 100× in buffer or water and used immediately, or diluted 80× in an aqueous glutaraldehyde solution (final aldehyde concentration, 0.1%) and used after 5 min of incubation at room temperature. For mass measurement, 7 μl aliquots were adsorbed for 1 min to glow-discharged thin carbon films supported on holey carbon films on 200-mesh gold-plated copper grids. The grids were blotted, washed in 7–12 drops of quartz double-distilled water, plunge-frozen in liquid nitrogen, and freeze-dried at − 80 °C and 5 × 10− 8 Torr overnight in the microscope. Tobacco mosaic virus (kindly supplied by Dr. R. Diaz-Avalos, Institute of Molecular Biophysics, Florida State University) adsorbed to a separate grid, was air-dried, and served as mass standard. A Vacuum Generators STEM HB-5 interfaced to a modular computer system (Tietz Video and Image Processing Systems GmbH, D-8035 Gauting, Germany) was used to record 512 × 512-pixel dark-field images. An accelerating voltage of 80 kV, a nominal magnification of 200,000×, and doses of approximately 400 e/nm2 were used for the mass measurements. Repeated low-dose scans were also recorded from some grid regions to assess beam-induced mass loss. The IMPSYS59 and MASDET60 program packages were used for evaluation. Fibril segments were selected in square boxes and tracked. The total scattering within an integration box following their length was then calculated, and the scattering contribution of the supporting carbon film was subtracted. Division by the segment length gave the MPL. MPL values were corrected for beam-induced mass loss, scaled to the MPL of tobacco mosaic virus, binned, displayed in histograms, and fitted by Gaussian curves. The measured MPL was not dependent on either the dilution method or the glutaraldehyde treatment, allowing all data sets to be merged and displayed in a single histogram for the final analysis.