Figure 3.

Noncontact AFM imaging of fibrinogen. (A) Tapping mode topographic image of fibrin monomers on mica 30 s after thrombin injection. (B) Noncontact topographic (left) and phase image (right) of an area offset 500 nm to the left of A, hence half the image has been imaged previously in tapping mode (within the dashed line), and half is imaged for the first time. An arrow pointing to the same group of molecules is included for guidance. This shows the irreversible damage to the soft molecules caused by imaging in tapping mode. The noncontact mode was unstable and switched repeatedly to H-state tapping (labeled 1–4). The phase image has been line leveled, and has a z-scale of 5°. Sections of the unleveled raw phase image are overlaid on the left and right edge (z-scale = 150°), in which dark contrast indicates noncontact mode, switching to tapping mode in the lighter contrast areas with a 70° shift in phase angle. Scale bar, 100 nm (1 μm images). (C) Zoomed 100 nm images of individual molecules together with overlaid line scans of three molecules, highlighting the flattened molecule in tapping mode (first column), the same molecules subsequently imaged in noncontact mode (second column), contrasted with molecules imaged in noncontact mode for the first time. (D–F) Presentation of a higher contrast inverted color scale to highlight αC regions. (D) Fibrinogen molecules, with no αC regions visible, contrasting with E and F that were taken 1 min after thrombin. αC regions were visible extending from most of the isolated molecules, tethering two molecules together, or extending from various points on the oligomers in E. αC regions were also visible in the noncontact molecules of C. Scale bar in c–e, 50 nm. The z-scale of 5 nm is the same in all topographic images.