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

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.

Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples

“…In the untreated control EP the typical type I collagen Raman bands are present at 852 cm −1 (Hydroxyproline), 938 cm −1 (C-C α stretch), 1255 cm −1 (Amide III), 1452 cm −1 (CH 2 collagen), and 1662 cm −1 (Amide I). 6 New Raman bands emerge at 1147 cm −1 and 1413 cm −1 , which can be assigned to the in-plane vibrations of the ring system of GE cross-links, at 1535 cm −1 (vibrations of the five membered ring of the GE cross-links) and 1718 cm −1 (CvO) upon GE cross-linking. 5 For clarity, collagen bands are only labeled in Fig.…”

“…[1][2][3][4] A good example of a strong fluorescent biological sample is equine pericardium (EP) after its cross-linking with Genipin (GE). 5 The pericardium of animals, which consists primarily of type I collagen and elastin, 6 is regularly used for tissue engineering as a scaffold for vascular grafts, patches, aortic valves, and wound healing procedures. To strengthen the pericardium structure and enhance its durability, cross-linking protocols have been developed.…”

Looking for a perfect match: multimodal combinations of Raman spectroscopy for biomedical applications

“…In a large-scale study, using four optical modalities SHG, two-photon fluorescence, FLIM, and RS, Shaik et al. 89 investigated the chemical, structural, and functional alterations in collagenous tissue upon incubation with collagenase and correlated the results with mechanical strength revealed by atomic force microscopy measurements. Figure 3 shows the time-dependent digestion of decellularized equine pericardium by bacterial collagenase with a clear shift toward lower fluorescence lifetimes and a distinct change in the spectral profile of corresponding Raman spectra.…”

“…(c) Mean normalized Raman spectra of three untreated (black) and digested (red) samples uncover that the lifetime shift correlates with increased elastin bands in the treated samples at 32 h. (d) A non-negative least-squares fitting algorithm reveals the reduction of collagen and increase of elastin for digested samples over time. Reprinted under a Creative Commons Attribution (CC-BY) 4.0 international license from Shaik et al 89 …”

Raman Spectroscopy in Skeletal Tissue Disorders and Tissue Engineering: Present and Prospective

“…However, as for cell characterization, methods to analyse matrix deposition are mainly invasive, long-term, and time-consuming. Thus, the strength of using RS in TE relies on the possibility to conduct sample analysis directly at the ECM biomolecules level in a non-invasive way.With the aim of monitoring changes in the properties of collagenous tissues, Shaik et al119 used several non-destructive techniques including Raman spectroscopy to monitor collagen digestion phases over time at the molecular level. Raman spectra intensity in the proline, hydroxyproline, C-Cα stretch and amide I region decreased over time.…”