Thermally Induced Irreversible Conformational Changes in Collagen Probed by Optical Second Harmonic Generation and Laser-induced Fluorescence

Abstract

Irreversible thermal conformational changes induced to collagen have been studied by optical methods. More specifically, second harmonic generation (SHG) from incident nanosecond Ng:YAG 1064 nm radiation and laser-induced fluorescence by 337 nm, pulsed nanosecond nitrogen laser excitation, at 405, 410 and 415 nm emission wavelengths were registered at eight temperatures (40 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees and 80 degrees C) and normalised with respect to the corresponding values at the ambient temperature of 30 degrees C. The heating protocol used in this work, was selected to monitor only permanent changes reflecting in the optical properties of the samples under investigation. In this context, the SHG, directly related to the collagen fibril population in triple helix conformation, indicated on irreversible phase transition around 64 degrees C. On the other hand fluorescence related to the destruction of cross-linked chromophores in collagen, some of which are related to the triple helix tertiary structure, also indicated a permanent phase transition around 63 degrees C. These results are in agreement with previous results from studies with differential scanning calorimetry. However SHG and fluorescence, being non-invasive optical methods are expected to have a significant impact in the fields of laser ablative surgery and laser tissue welding.

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…In particular, SHG microscopy has been demonstrated extremely powerful to image collagen rich tissues [65] such as cornea [66,67], tendon [68,69], and arteries [70]. In particular, SHG microscopy has been mainly used for selectively investigating collagen fibres orientation and their structural changes in human dermis [71][72][73][74][75], keloid [76][77][78], fibrosis [79][80][81], thermally-treated samples [82][83][84][85][86], and also in tumour microenvironments [87][88][89][90][91][92][93]. In fact, SHG microscopy highlights morphologic changes in collagen structure, which indicate particular disease states, such as tumour invasiveness, as well as indicators of collagen remodelling in tumour stroma, which is playing a key-role in the tumour development from in-situ to invasive stage.…”

Laser-assisted vessel welding: state of the art and future outlook

“…The different laser wavelengths, lasing parameters, and tissue molecular compositions produce different thermodynamic profiles. Heath-induced collagen denaturation starts at a temperature of ~60 °C and becomes irreversible when tissue reaches a temperature of ~64 °C [79]. Thus, if lasing is terminated at around 70-75 °C, welding is achieved by collagen cross-linking.…”

Protein conformation and molecular order probed by second-harmonic-generation microscopy

“…28,29 Collagen type I features a hierarchical organization ranging from the atomic and molecular scale (tropo-collagen molecules: 280 nm length and 1.5 nm diameter), to the microscopic scale (fibrils: 1 μm length and 30 nm diameter) and the macroscopic scale (fiber bundles). The sensitivity of SHG to these different hierarchical organizations allows probing the thermally induced structural changes of collagen [52][53][54][55] as shown in Fig. 4.…”