Figure 3.

Stretching NM fibrils by optical trapping at high forces with simultaneous fluorescent imaging. (a) Schematic of the trapping instrument with dual trapping capabilities used for high force experiments (see Methods for details). 1024, nm, trapping laser; 975 nm, position detection laser; 532 nm, fluorescence imaging. (b) Schematic representation and fluorescence snapshots of the experiments. From left to right, the bead was first centered on top of the attachment point, and then the stage was moved along the x-axis until the fibril was fully extended. (c) Representative pulling and relaxation traces for NM fibrils in the presence of normal assembly buffer. The force and the position of the stage were plotted as a function of time. Black line, the stretching and holding phase; red line, the relaxation phase; blue line, position of the bead relative to the attachment point of the tether on the glass surface. (d) Left, force applied vs. fibril extension length (force-extension curve) for the tethered fibril experiencing one stretching-relaxation cycle. Black line, stretching and holding phase; red line, relaxation phase. The x-axis is expanded to illustrate the behavior in the fully extended position. Right, the force-extension curves for a single tethered fibril that experienced four sequential stretching-relaxation cycles superimpose upon each other. For clarity, the holding phase is not shown. Stretching and relaxation curves from the same cycle are shown in the same color, with subsequent curves in a different color.