Wavelength-Dependent Conformational Changes of Collagen in Mid-IR Ablation

Single pulses of the Mark-III free electron laser have been used to ablate porcine corneas at a fluence of 240 J/cm$^{2}$ and wavelengths of 2.77 and 6.45 $\mu $m. As previously characterized, the non-volatile ablation debris shows evidence of wavelength-dependent collagen fragmentation. We have measured micro-Raman spectra of the debris and the ablation crater to determine if any wavelength-dependent conformational changes have taken place. Comparison of the spectra from two different wavelengths shows that a 938 cm$^{-1}$ Raman band -- assignable to the peptide C$_{C=O}$-C$_{\alpha }$ stretch of collagen -- loses substantial intensity during 6.45-$\mu $m ablation, but not in 2.77-$\mu $m ablation. This intensity decrease may be associated with a reduction of collagen triple-helix structure. Other spectral techniques yield mixed results; signatures for the loss of triple-helix structure are evident in UV-CD and some aspects of $^{13}$C-NMR spectra, but not in FTIR spectra. Raman measurements on thermally-treated corneal slices display similar changes at high temperatures, suggesting that higher protein temperatures are reached during ablation at 6.45 $\mu $m when compared to 2.77 $\mu $m. These observations suggest that any pre-vaporization loss of protein structural integrity includes not only collagen fragmentation, but also a loss of collagen triple-helix structure.