Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques

Year: 2021

Authors: Shaik T.A., Lagarto J.L., Baria E., Goktas M., Onoja P.I., Blank K.G., Pavone F.S., Popp J., Krafft C., Cicchi R.,

Autors Affiliation: Leibniz Institute of Photonic Technology, 07745 Jena, Germany;
National Institute of Optics (INO), National Research Council (CNR), 50125 Florence, Italy;
European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, 50019 Sesto Fiorentino, Italy;
Mechano(bio)chemistry, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany;
Friedrich Schiller University, 07743 Jena, Germany;
Abbe Center of Photonics, Friedrich Schiller University, 07745 Jena, Germany;

Abstract: We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 μg/mL bacterial collagenase at 37  C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm−1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young’s modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.


Volume: 93 (8)      Pages from: 3813  to: 3821

More Information: Horizon 2020 Framework Programme, Grant/Award Number: 654148 and 871124, (Laser Lab Europe), Horizon 2020 Framework Programme, Grant/Award Number: 732111 (PICCOLO), and Tuscany Region Program POR FSE 2014-2020 Giovanisi.
KeyWords: Raman spectroscopy; pericardium; scattering; cartilage; markers; signal; collagen; tissue engineering
DOI: 10.1021/acs.analchem.0c04306

Citations: 10
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