Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

Abstract

The non-destructive and label-free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non-contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400-1800 cm(-1)), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof-of-principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically-degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non-destructive and non-contact tool to probe ECM state in situ.

Molecular pathology via IR and Raman spectral imaging

“…Furthermore, it has the enormous advantage of relative immunity from water interference; thus it is readily amenable to data collection of live tissue or in-vivo applications. In fact, several research groups have published numerous reports on Raman histopathology of hollow organs or extracellular matrix [29], or acquired through needles fitted with fiber optics [30][31][32][33]. Although the spatial resolution of these methods is less than that of microscopic Ra-SHP, the fact that an entire tissue fingerprint can be collected in a few seconds of data acquisition time bears an enormous advantage for in-vivo tissue diagnosis.…”

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

“…13 We also identified that changes in the overall Raman signal intensity can serve as an indicator for the state of collagen and collagen fibers within the ECM of aortic valve leaflets. 9 However, when analyzing the state of elastin in these tissues, we were not able to detect similar results. As mentioned in the results section, we were only able to detect alterations of specific Raman bands in the elastase-treated samples when compared to the native controls.…”

“…These observations resulted in a score plot that did not reveal a clear cluster formation as seen in the previous study. 9 In contrast, the influence of the enzymatic treatment was detectable within the PCA results. We assume that these discrepancies between the two ECM proteins, elastin and collagen, are based on morphological differences and different enzymatic degradation processes: within the aortic valve leaflet, the collagen-rich zone (fibrosa) is a continuous layer that becomes loosened due to the enzymatic treatment, whereby the elastin containing zone (ventricularis) has a network configuration that appears fragmented after exposure to elastase (Fig.…”

Continuum theory of fibrous tissue damage mechanics using bond kinetics: application to cartilage tissue engineering

“…Therefore, modern measurement techniques that can directly detect chemical bond modifications and disruptions in biological tissues, such as biochemical measurements for assessing cross-link density in collagenous tissues [13], immunohistochemistry for assessing mechanically or enzymatically degraded collagens [14][15][16][17][18][19], and Raman spectroscopy for detecting alterations in chemical bonds [20,21], may not inform the traditional damage framework directly.…”

“…where z GAG is the charge number and M GAG is the molar mass of GAG (z GAG ¼ 22 and M GAG ¼ 513 g mol 21 , recognizing that GAG content was measured using a dimethylmethylene blue assay with chondroitin sulfate as the standard); r T is the construct mass density (taken to be r T ¼ 1 g ml…”