Multimodal Nonlinear Vibrational Hyperspectral Imaging

Linescanning vibrational sum-frequency generation (VSFG) hyperspectral microscopy was developed into an inverted microscope design. The geometry enables seamless collection of brightfield, second-harmonic generation (SHG), and VSFG images of a given sample area. The new vertical configuration also enables future application to biologically relevant environments. The instrument is capable of simultaneously reporting on spatially resolved chemical and geometric specific sample characteristics. This capability is demonstrated with three samples: lyophilized collagen, a molecular self-assembly of sodium dodecyl sulfate and -cyclodextrin (SDS@2-BCD), and a L-phenylalanyl-L-phenylalanine (FF) self-assembly. Hyperspectral analysis showed that the FF samples have anisotropic structural alignment, which is uniform along the long axis and structurally evolving along the short radial axis. Because all three samples represent protein and molecular hierarchically organized materials in the biomaterial and biomimetic fields, this work highlights the chemical-physical information VSFG microscopy can reveal to help in the bottom-up design and characterization of biomaterials.