We demonstrate the capability of linear and non-linear optical microspectroscopy techniques such as fluorescence lifetime imaging (FLIM), Raman spectroscopy (RS), second-harmonic generation (SHG), and two-photon fluorescence (TPF), to monitor structural, biochemical and biomechanical alterations in collagenous tissues upon both collagen digestion and cross-linking using genipin as cross-linker. Thanks to a unique combination of optical and force microscopy techniques in a correlative manner, we obtained detailed information about the biochemical, structural, and biomechanical properties of collagenous tissues upon both digestion and cross-linking treatment.
Genipin cross-linked engineered tissues are 10000 times less toxic than glutaraldehyde cross-linked tissues. Hence, genipin is a better fixative to support the recellularization of tissue-engineered constructs such as vascular grafts. Here, we demonstrate the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of genipin cross-linked vascular grafts with high speed and specificity.
Current results indicate that the fluorescence lifetime of AR-BP shortens upon GE cross-linking. Raman spectroscopy reveals secondary structural changes occurring in the extracellular matrix of pericardia that correspond to Amide I, Amide III and C-C stretch vibrations.
We conclude that FLIm guided Raman imaging can detect cross-linking signatures with biochemical specificity and that this imaging modality provides a non-destructive and label-free method to assess the quality of vascular grafts
We demonstrated the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of engineered vascular grafts with high speed and specificity. We report FLIm guided Raman imaging as an effective multimodal technique to evaluate scaffold cross-linking and localized calcification. Current results indicate that the lifetime of AR-BP shortens upon GA cross-linking,and Raman spectroscopy reveals secondary structural changes occurring in the Amide I region of cross-linked pericardia. GA fixed vascular grafts are prone to calcification, an effect linked to graft failure. The calcified regions exhibited shorter lifetimes in fluorescence spectral bands ranging from 380 to 455 nm and Raman spectra exhibited the specific hydroxyapatite signature at 960 cm-1 co-localized with these lower lifetime regions. We conclude that FLIm guided Raman imaging can detect cross-linking signatures and areas of calcification in tissue with biochemical specificity.
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