relaxed or prestrained rectangular silicone sheets. The ﬁbers were then rinsed four times and then equilibrated in a phosphate buffered saline (PBS) solution and then subse- quently strained or relaxed to deﬁned amounts in a custom- built one-dimensional strain device at room temperature described previously.14 Colocalization Analysis of Protein Binding to Fibrillar Fn. In order to quantitatively describe the relationship between the binding of a protein from solution to the conformation of ﬁbrillar Fn, samples of manually deposited Fn ﬁbers were prepared as described above. BSA, casein, and the 70 kDa Fn fragment were acquired from Sigma, and along with the isolated Fn described above, both proteins were labeled with Alexa Fluor 633 ﬂuorophores on random amines following the manufacture’s protocol. Each protein was incubated with the samples for 10 min at room temperature (at solution concentrations: BSA and casein, 100 µ g/mL; Fn, 3 µ g/mL; 70 kD Fn, 1 µ g/mL) and rinsed with a PBS or 2% BSA solution before imaging. Because the z-resolution of the confocal microscope was 1 µ m, the middle slice of the ﬁbers was taken for all data analysis. For all adsorbed surfaces, we observed that the ﬁber interior was brightly ﬂuorescent as well, indicating that the interior is accessible to soluble proteins. Experiments were also re- peated with widely varying concentrations and incubation times, and the results did not change (data not shown). Furthermore, to block nonspeciﬁc adsorption, samples with casein and the 70 kDa Fn fragment were preincubated with a 2% BSA solution for 1 h before introduction of the labeled protein. To block speciﬁc adsorption and test for nonspeciﬁc adsorption alone, samples of Fn were incubated with a 100× more concentrated solution of unlabeled protein before introduction of the labeled protein. In addition, the mouse antihuman L8 monoclonal antibody, received as a generous gift from Michael Chernousov, was adsorbed at a 1:200 dilution for 10 min to samples blocked for 1 h with a 2% BSA solution before rinsing with 2% BSA and incubating for another 10 min with a 1:200 dilution of a Cy5 donkey antimouse secondary antibody (Jackson Antibody). As a control, incubation with the Cy5 secondary antibody alone was performed, and no background adsorption to the ﬁbers was observed (data not shown). Fn Refolding and Release of Adsorbed Proteins. To study refolding kinetics of ﬁbrillar Fn and the release of bound proteins, 5% Fn-DA ﬁbers were manually deposited in parallel orientations onto prestrained substrates, rinsed four times with PBS, and incubated with BSA-633 (at 100 µ g/ mL) or nothing as a control. After imaging the ﬁber samples in an initial absolute 140 or 300% strain (average IA/ID of 0.67 and 0.62, respectively; Figure 3a), the ﬁbers were relaxed to 0 (“fully relaxed”) or 60% strain. Then, we imaged again at 5, 10, 20, and 30 min. After this point, the samples were again stretched to their initial conformations and imaged a ﬁnal time at the same original ﬁeld of view. In addition to recording the average IA/ID values of individual ﬁbers at each time and position point, the intensity of each far-red channel pixel (633 nm excitation, 645-745 nm detection) was normalized by the intensity of the acceptor channel only (546 Nano Lett., Vol. 9, No. 12, 2009 nm excitation, 566-578 nm detection). Because the intensity of the substrate Fn increased signiﬁcantly upon ﬁber relaxation, the donor channel could not be kept constant or used in the normalization analysis without saturating the PMT. The average ratio of the intensity of bound protein to the intensity of the directly excited acceptor for each pixel comprising a ﬁber was then plotted as in Figure 3b. Bis-ANS Binding and Analysis. Fn ﬁbers were manually deposited from a 0.3 g/L solution of 10% Alexa Fluor 633- labeled Fn (Fn-633) onto prestrained silicone substrates in a “star”-like pattern and, upon full relaxation of the substrate (0% strain), produced a population of Fn known to have a fully relaxed conformation and another with a strained conformation (300% strain; Supporting Information, Figure S2A). To detect the exposure of hydrophobic patches on the intact secondary protein structure, a 0.4 mM solution of bis- ANS was incubated for 45 min on the ﬁber samples and rinsed four times with PBS. Experiments were also repeated with 1 h or 10 min incubation times, and the results did not change. Images were acquired by exiting the samples with laser wavelengths of 454 and 633 nm while collecting emitted light between 475-575 nm (Channel 1) and 645-745 nm (Channel 2). Using a custom script written in Ruby (www.ruby-lang.org) together with image analysis software (ImageMagick Software Suite, www.imagemagick.org), the background from each channel was subtracted and a 1 pixel radius Gaussian blur (with a 1 pixel standard deviation) was applied to smooth errant intensity values before the intensity of the bis-ANS channel was divided by the intensity of the corresponding Fn-633 channel. This ratio was then mapped to false shades of blue, as shown in Figure 2j and plotted in the histogram. To determine the strain factor (l) of ﬁbers deposited at an angle (θ) to the stretch axis of substrates relaxed by a factor of ε with a transverse extension factor of εT, the following equation was used l ) (( ε ) + ( εT ) ) cos θ 2 sin θ 2 -1/2 Iodocetamide Experiments. To further investigate the exposure of hydrophobic residues, we compared Fn ﬁbers pulled from a labeled Fn solution with ﬁbers pulled out of a solution where the hydrophobic residues were already alkylated in denaturant. Fibers were pulled from a drop of Fn treated with iodoacetamide, which oxidizes free sulphides to prevent maleimide dye binding, in the presence of denaturant. Then, the drop of alkylated Fn was removed. A drop of wild-type human serum Fn labeled with Alexa 633 on lysines was positioned at the opposite side, and ﬁbers were pulled and deposited in between ﬁbers of alkylated Fn. The drop of Alexa 633 Fn was removed. After washing the sample, the substrate was stretched under 4% BSA. Finally, 5 ng of Alexa 488 maleimide was added to the solution and incubated for 15 min to investigate where/if it is able to bind to the newly exposed hydrophobic patches. In a second set of experiments, we investigated 633-labeled Fn ﬁbers deposited on polydimethyl- siloxan (PDMS) sheets as before in different starting positions (star shape) but then blocked the already exposed hydrophobic patches on these ﬁbers with iodoacetamide. After we applied 4165