## 2 Materials and Methods
### Imaging Collagen-Binding Peptides with Quantum Dots on Rat-Tail Type I Collagen  
#### 2.2.1 Peptide Synthesis  
TKKTLRT and TKKLTLRT peptides were synthesized on RINK amide resin (RFT105, AAPPTIEC, Louisville, KY, USA) using the AAPPTIEC Focus XC synthesizer (Louisville, KY, USA) with standard Fmoc solid-phase peptide synthesis protocol. Post-synthesis, the peptides were biotinylated, taking advantage of avidin-biotin chemistry for running collagen-binding assays using streptavidin-conjugated quantum dots. Peptide biotinylation followed the AAPPTIec protocol for activation and coupling of D-biotin (AAPPTIEC, Louisville, KY, USA). The peptide was suspended in dimethylformamide (DMF, 9.8%, Sigma-Aldrich, St. Louis, MO, USA) at a volume of 10 mL per gram of resin-bound peptide. Separately, 1 mmol of biotin per 1 mL solution of dimethyl sulfoxide (DMSO, 99.7%, Sigma-Aldrich, St. Louis, MO, USA) and DMF (1:1 \(v/v\)) is prepared. One equivalent of hexafluorophosphate (HBTU, AAPPTIEC, Louisville, KY, USA) and \(N\)\(N^{\prime}\)-Disisopropylthelylamine (DIEA, 99.5% nitrogen flushed, Acros Organics, Fair Lawn, NJ, USA), respectively, were added to the mixture and stirred at room temperature until the biotin was fully dissolved. Once dissolved, the resin-bound peptide solution was added and mixed at room temperature for three hours. After biotinylation was complete, the peptides were filtered, washed with ethanol, dried, and ready for cleavage. Peptide cleavage followed the AAPPTEC protocol. The cleavage cocktail of the peptides contained trifluoroacetic acid (TFA, 99%, Sigma-Aldrich, St. Louis, MO, USA), thioanisole (99%, Sigma-Aldrich, St. Louis, MO, USA), 1,2-ethanedithiol (95%, Acros Organics, Fair Lawn, NJ, USA), trisopropylsilane (98%, Sigma-Aldrich, St. Louis, MO, USA), and DI water at the following ratios: 87.5:5.0.2.5.2.5.2.5. The peptides were left in the cleavage cocktail on a rotator for 2 h, subsequently precipitated in cold ether, and lyophilized.  
Peptide purification was initially performed on the semi-preparative reverse-phase high-pressure liquid chromatography (HPLC) Waters system, with the Waters 600 controller and Waters 2487 Dual Absorbance Detector. The system used the 10 um C-18 silica Luna column (250 \(\times\) 10 mm, Phenomenex Inc., Torrance, CA, USA). The mobile phase was comprised of phase A (94.5% HPLC grade water, 5% acetonitrile, 0.5% TFA) and phase B (100% acetonitrile). lyophilized peptides were dissolved in 4 mL of phase A then purified on a linear gradient at 0.5% phase B-min\({}^{-1}\) (5%-85% phase B) with a flow rate of 3 mL-min\({}^{-1}\) with detection at 254 nm, which was conducted at room temperature. The resulting purified peptide fractions collected were verified using the analytical Shimadzu HPLC system, an LC-2010 HT liquid chromatograph, and an SPD-M20A prominence diode array detector. A 5 um C-18 silica Luna column (250 \(\times\) 4.6 mm, Phenomenex Inc., Torrance, CA, USA) was used with a mobile phase composed of phase A (99.9% HPLC-grade water, 0.1% TFA) and phase B (100% acetonitrile). The system ran on a linear gradient with 1 mL-min\({}^{-1}\) flow, 40 \({}^{\circ}\)C, and 254 nm detection. The purified peptides were lyophilized and stored at \(-\)20 \({}^{\circ}\)C.