Next, to characterize the aggregation process further, we performed time-lapse AFM imaging experiments in which images were taken over the same surface area continuously after injecting 100 nM Aβ-42 protein in 10 mM phosphate buffer (pH 7.4) into the fluid cell. AFM images for selected observation times are shown in Supplementary Fig. 2A. Frame (i) shows the image of the surface prior to adding the protein, illustrating that the surface is clean. The next frame (ii) shows the image taken after 10 min, and here again no protein aggregates are seen. As the incubation time increases, new aggregates appear on the surface. For example, frame (iii) illustrates that quite a large number of aggregates appear after 5 h incubation. Comparison with the images taken after 6 h demonstrates the dynamics of the aggregation process in which aggregates are formed and dissociate and grow. For example, an aggregate circled in yellow in frame (iii) is no longer present in (iv), and the aggregate circled in yellow in (iv) disappears in (v), suggesting that these aggregates have dissociated from the surface. At the same time, a new aggregate circled in green appears in (iv). The aggregate indicated by a dotted black circle remains throughout the time-period of imaging (Supplementary Fig. 2iii–vii) and serves as a marker to monitor the changes occurring in other aggregates. The aggregates marked with a yellow dotted circle dissociate from the surface, whereas the new aggregates which appear on the surface are indicated with a green dotted circle. The results are summarized graphically in Supplementary Fig. 2B. In this graph, the time-dependent changes of overall volume and number of aggregates are plotted. The graphs demonstrate that the particle volumes increase over time monotonically, although the number of aggregates is not monotonic, indicating that aggregation occurs as a dynamic process in which aggregates can dissociate from the surface. To illustrate these dynamics for individual aggregates, we numbered a few particles as shown in Supplementary Fig. 2 iii-vii. For example, the aggregate marked ‘1’ stays in plates iii and iv (Supplementary Fig. 2A) but disappears in plate v. The aggregate marked ‘2’ shows up in plate iv and disappears in plate vii (Supplementary Fig. 2A). Measurement of the aggregate sizes (Supplementary Table 1) demonstrated that some of the aggregates grow in size leading to an increase in volume (aggregates ‘3’, ‘4’, ‘5’ and ‘6’), whereas some dissociate from the surface (aggregates ‘1’ and ‘2’). To estimate the subunit numbers of the oligomers, we compared the volumes determined here with volumes of isolated Aβ-42 oligomers of specific sizes obtained by photoinduced cross-linking22. These measurements (data not shown) show that the heptamer and decamer have volumes of 60.3 ± 9.7 nm3 and 73.4 ± 12.8 nm3 respectively, suggesting that the oligomers formed after 8 h of the time-lapse experiment correspond to the oligomer order of heptamer to decamer.