Fluorescence (A, D, G, J, M) and SHG (B, E, H, K, N) images collected simultaneously from (A, B) Silk fibers, (D, E) a methanol-treated aqueous film, (G,H) a compressed and stretched methanol-treated aqueous film, (J,K) a hydrogel, and (M,N) a silk scaffold. All images were acquired with 800nm excitation through a 20× (0.7NA) objective. Fluorescence was collected through a 525nm filter with a 25nm band pass. SHG was collected in the forward direction through a 410nm filter with a 20nm band pass. Some of the main secondary structure features, including b sheet content and alignment are depicted for (C) silk fibers, (F) methanol treated films, (I) compressed methanol treated films, (L) hydrogels, and (O) scaffolds. (Bar A-GM =100μm, Bar J = 20μm)