Wavelength-dependent conformational changes in collagen after mid-infrared laser ablation of cornea

Abstract

We ablated porcine corneas with a free electron laser tuned to either 2.77 or 6.45 microm, two matched wavelengths that predominantly target water and protein, respectively. The ejected nonvolatile debris and the crater left behind were examined by circular dichroism, Raman spectroscopy, and scanning electron microscopy to characterize the postablation conformation of collagen proteins. We found near-complete unfolding of collagen secondary and tertiary structure at either ablating wavelength. On the other hand, we found excess fibril swelling and evidence for excess cis-hydroxyproline in the 6.45-microm debris. These results support the hypothesis that the favorable ablative properties of protein-targeting wavelengths rest on selective heating of tissue proteins.

FIGURE 1

VCD spectra of debris collected from ablation of porcine corneas at a wavelength of either 2.77 (dashed lines) or 6.45 μm (dotted lines). The debris spectra were measured in situ on a CaF₂ window. The VCD spectrum of a dried slice of cornea is shown for comparison (solid line). The inset contains the corresponding infrared absorption spectra. Vertical bars mark the scale for each set of spectra in absorbance units.

FIGURE 2

UVCD spectra of debris collected from ablation of porcine corneas at a wavelength of either 2.77 (dashed lines) or 6.45 μm (dotted lines). The debris was resolubilized in distilled water. UVCD spectra of a purified collagen solution at 30°, 40°, and 50°C (solid lines) are shown for comparison. The magnitude of the UVCD signals decreases as the collagen triple helices unfold at higher temperatures.

FIGURE 3

Scanning electron micrographs of collagen fibrils in (a) slices of unexposed cornea, (b) the debris field from 2.77-μm ablation, and (c) the debris field from 6.45-μm ablation. The horizontal scale bar is applicable to all three images.

FIGURE 4

Raman spectra of the ablation debris and selected locations in and around the ablation craters. The ablating wavelength was either (a) 2.77 μm or (b) 6.45 μm. In each panel, the upper spectra are from the locations marked in the inset brightfield images. The dotted shaded curve is from outside the crater (solid arrow). The solid curves are from positions on the rim, side, and bottom of the crater (open arrows). The lower spectrum in each panel is from the ablation debris. The Raman spectra have all been normalized by the maximum intensity of the 920 cm⁻¹ band. The normalized level for each spectrum is marked by a horizontal dashed line. The inset brightfield images are from unstained corneas. The horizontal scale bar (20 μm) is applicable to both images.

FIGURE 5

Temperature dependence of the 938 cm⁻¹ Raman band in dried corneal slices. (a) Spectra measured at elevated temperatures of 60°, 100°, 150°, and 200°C; and (b) spectra measured after such heating and reequilibration at room temperature. The Raman spectra have all been normalized by the maximum intensity of the 920 cm⁻¹ band. This normalized level is marked by the horizontal dashed line. The dotted shaded curve in each panel is from a sample dried at room temperature and never heated. The solid curves are from the heated samples. The intensity at 938 cm⁻¹ is largest in the unheated sample and decreases further with each increase in sample temperature.

FIGURE 6

Effect of salt concentration on (hydroxy)proline isomerization. (a and b) Raman spectra are shown for films dried down from a collagen solution in either (a) distilled water or (b) 0.1 M NaCl. (c and d) ¹³C-NMR spectra are shown for collagen solutions in DMSO/water (70:30 v/v) with (c) no added salt or (d) 0.3 M NaCl. The marked NMR resonances are for trans- (70 ppm) and cis-hydroxyproline (68 ppm) (14).