Figure 4. Colocalization analysis of soluble Fn and the 70 kDa Fn fragment binding to stretched or relaxed ﬁbrillar Fn. The data were analyzed and displayed analogous to the ones shown in Figure 1. (a-d) Full-length Fn and (e-h) is its 70 kD N-terminal fragment. Methods. Isolation and Fluorescent Labeling of Fi- bronectin. Fn was isolated from human plasma (Swiss Red Cross) via a previously described procedure10,14,51 and dual- labeled (Fn-DA) with Alexa Fluor 488 succinimidyl ester and Alexa Fluor 546 maleimide for FRET experiments, also as previously described.14 The eluant solution from the ﬁnal step of the labeling procedure was determined via spectro- photography to have Fn labeled on average with 6.0 donors (Alexa Fluor 488) and 3.6 acceptors (Alexa Fluor 546). This batch of Fn-DA, which was the only one used to collect data for this report, was stored in frozen 10 µ L aliquots and only used when thawed for less than 5 days. 4164 Mechanical Strain and Chemical Unfolding Calibra- tions, Microscopy, and FRET Analysis. The batch of soluble Fn-DA used in this report was ﬁrst calibrated for chemically induced unfolding in increasing GdnHCl solutions as described previously.14 Images were acquired via scanning laser confocal microscopy (Olympus FV-1000), and the degree of intramolecular FRET was probed pixel-by-pixel by dividing the intensity of the acceptor channel by the intensity of the donor channel (IA/ID), as previously de- scribed.14 To establish an IA/ID versus mechanical strain calibration curve, a 0.3 g/L solution of 95% unlabeled Fn and 5% Fn-DA was used to manually deposit Fn ﬁbers onto Nano Lett., Vol. 9, No. 12, 2009