uniaxial stretch to the substrate and therewith stretched or orthogonally relaxed the ﬁbers, we added Alexa 488 maleimide. Data analysis was performed by calculating the Alexa 488 to 633 ﬂuorescence ratios. Image Analysis. To determine FRET from Fn ﬁbrils, the ﬁeld of view was excited with 488 nm light, and the respective ﬂuorescence intensities were determined for the donor channel (514-526 nm) and acceptor channel (566-578 nm), pixel-by-pixel, to determine IA/ID when excited at 488 nm or 633 nm, respectively. For colocal- ization analyses, a third image of the same ﬁeld of view was acquired in the far-red channel (645-745 nm) with 633 nm excitation light to detect the intensity of the adsorbed protein. Since ﬁber diameters are random and also change dramatically with strain, we next sought to normalize the intensity of the ﬂuorescent, soluble binding receptor with the mass of Fn in each pixel. This normal- ized colocalization data was acquired using a custom script written with Ruby and Imagemagick to produce contour plots of the normalized protein intensity. Brieﬂy, in addition to the handling of the data to produce the IA/ID ratios, histograms, and false color image maps as described previously,14 the script also background subtracted and applied a 1 pixel radius Gaussian blur (with a 1 pixel standard deviation) to the far-red channel. In order to normalize for stretch-related changes in Fn density, the emission intensity of Alexa 633-BSA to the amount of Fn present in each pixel was divided by the sum of the corresponding donor and acceptor channel intensities (IA + ID). Although this analysis assumes that the quantum efﬁciency of detecting emission from the donor and acceptor is equivalent, this analysis was similar to a normalization scheme where IAlexa633 was divided by the intensity of the acceptor ﬂuorophore but with direct 543 nm excitation. With direct excitation of Alexa 546, energy transfer as well as crosstalk from Alexa 488 into the Alexa 546 emission window was minimal. However, in order to reduce the number of images acquired with each sample, the sum of IA + ID was used for all analysis in the present study. From the same ﬁeld of view as the raw protein-633 channel (Figure 1c), the donor and acceptor channels were acquired, and the standard spatially resolved FRET analysis was performed to produce IA/ID histograms (Figure 1b), as described previously.10,14 Overlaid on the contour plot in Figure 1d between the area corresponding to the fully relaxed and ﬁber breakage zone conformations is the median normalized intensity for the corresponding IA/ID bin from the contour plot of the same data as shown in Figure 1h. Acknowledgment. The authors gratefully acknowledge Kristopher Kubow and John Saeger for their helpful discus- sions and Sheila Luna Morris for her help in preparing the ﬁgures. This work was ﬁnancially supported by the ETH Zurich, the Human Frontier Science Program Organization (M.L.S.), the Volkswagen Stiftung, the Nanomedicine De- velopment Center (NDC) “Nanotechnology Center for Mechanics in Regenerative Medicine” (NIH Grant PN2 EY016586), which is part of the NIH Nanomedicine Development Center Network (NNDCN), and an Advanced 4166 Investigator Award for Interdisciplinary Research from the European Research Council (V.V.). The mouse antihuman L8 monoclonal antibody was a generous gift from Dr. Michael Chemousov. The authors declare no competing ﬁnancial interests. Supporting Information Available: Additional ﬁgures. This material is available free of charge via the Internet at http://pubs.acs.org. Abbreviations ECM, extracellular matrix Fn, ﬁbronectin FRET, ﬂuorescence resonance energy transfer Fn-DA, Fn labeled with donor and acceptor ﬂuorophores GdnHCL, guanidine hydrochloride IA/ID, ratio of the acceptor intensity divided by the donor intensity bis-ANS, 4,4′-dianilino-1,1′-binaphthyl- 5,5′-disulfonic acid, dipotassium salt L8, monoclonal Fn antibody (epitope: residues 526-675 span- ning FnI9 and FnIII1) BSA, bovine serum albumin PBS, phosphate buffered saline References (1) Kellermayer, M. S.; Smith, S. B.; Granzier, H. L.; Bustamante, C. Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 1997, 276, 1112–1116. (2) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 1997, 276, 1109–1112. (3) Tskhovrebova, L.; Trinick, J. Titin: properties and family relationships. Nat. ReV. Mol. Cell Biol. 2003, 4, 679–689. 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