1.

Introduction

The ability to monitor corneal structural changes is important in many ophthalmologic applications. Surgical procedures such as conductive keratoplasty and laser thermokeratoplasty depend on the structural alteration of corneal structure in achieving vision correction.^{1, 2} Since the cornea is a highly transparent object, the development of an effective imaging technique to obtain both qualitative and quantitative information of corneal structural changes without histological or labeling procedures can be a useful technology in guiding surgery and other therapeutic procedures in ophthalmology. Presently, histological techniques remain the most commonly used imaging platform in characterizing corneal changes.^{3, 4, 5, 6} However, the development of nonlinear optical microscopic techniques offers an exciting imaging modality capable of circumventing the drawbacks of conventional histological procedures. While second harmonic generation (SHG) was used to image crystalline structures in the 1970s, the biomedical applications of nonlinear optical microscopy flourished with the introduction of two-photon fluorescence microscopy pioneered by Webb’s group in 1990.^{7, 8, 9} There are a number of important advantages in the applications of nonlinear optical microscopy. First, since the excitation probability is proportional to the square of excitation intensity, sample excitation only occurs effectively at the focal point. Therefore, microscopic imaging with this technique results in images with excellent axial-depth discrimination without using confocal detection. In addition, since photodamage only occurs at the focal point, the overall specimen damage is greatly reduced. Finally, since the near-infrared laser sources used in multiphoton microscopy are absorbed and scattered less than the ultraviolet or visible photons used in one-photon excitation microscopy, multiphoton imaging allows greater sample depths to be achieved without histological procedures.¹⁰

In harmonic generation imaging, the nonlinear polarization effect at a high electric field can be used to explore a sample’s microscopic structures. In general, the polarization $P$ of a material can be expressed as

2.

Methods

3.

Results

Using SHG microscopy, we acquired large area scans of the porcine corneas treated for 5 min at different temperatures from 25 to 90°C. Shown in Fig. 1 are a representative set of the cross-sectional SHG images of the cornea stroma of 18 temperatures at 25, 30, 35, 40, 45, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 85, and 90°C. In each panel, the SHG images (left figures) are presented using the displayed color scale for indicating the minimum and maximum intensities used for display. For comparison, the corresponding HE images at each temperature are shown (right figures) for comparison.

Fig. 1

Large area second harmonic images (left figures: $880\times 880\mu {\mathrm{m}}^2$ ) and HE images of porcine cornea after 5 min treatment at the respective temperatures.

In this manner, similar comparison between the SHG and HE images can be made. Visual examination of Fig. 1 reveals several important features. First, the collagen fibers associated with cornea stroma are clearly visible under SHG imaging. Furthermore, the SHG intensity changes as a function of temperature. Visual examination of the SHG images in Fig. 1 shows that with increasing temperature, distinct changes in SHG intensity occur. From 25 to 45°C, SHG intensity appears to decrease with increasing temperature. However, starting at 50°C, the SHG intensity increases and reaches maximum values between 50 and 71°C. With additional increase in temperature, SHG intensity starts to decrease. However, an additional maximum in SHG signal is observed at around 74 to 77°C. As the temperature continues to increase, the SHG signal decreases until 90°C, where the SHG intensity has reached a baseline value. The corresponding histological images show that at temperatures less than 53°C, the stroma collagen are organized into visible fibers. From 56 to 74°C, regions of the cornea appear to have been grouped into forming separate domains without the finer fibrous structures visible at lower temperatures. The appearance of cavities is also apparent at these higher temperatures. At temperatures higher than 77°C, the number of distinct domains decreases within the cornea. At 90°C, the histological image shows that the corneal collagen has merged into a continuum distribution of mass with no discernible microscopic features. To add statistical significance to our data, the intensity from the SHG images from two additional porcine eyes were measured and averaged. The statistical analysis of SHG intensity from the set of three porcine eyes at each temperature is shown in Fig. 2 .

Fig. 2

Second harmonic intensity of porcine cornea stroma as a function of treatment temperature (5 min).

The plot in Fig. 2 shows that at 25 and 30°C, the SHG intensities are essentially the same (within experimental error). In the higher temperature range between 35 and 45°C, SHG intensity decreased. As the temperature further increases, the SHG intensity first increases to a peak at 53°C before reaching a main peak at 65°C. With additional increase in temperature, the SHG signal first decreased and then increased to a secondary peak at around 74 to 77°C. As the temperature continues to increase, the SHG intensity monotonically decreases to the background intensity at 90°C. Note that these observations are difficult to confirm from the histological images alone. The sharp rise in SHG intensity at 65°C can be attributed to a sharp, irreversible structural change of the cornea stroma at that temperature.

4.

Discussion

By applying SHG imaging to thermally treated cornea specimens in intact porcine eyes, we have shown that the collagen fibers of cornea stroma undergo four distinct structural changes in the temperature range between 25 to 90°C. First, between 30 and 45°C, the cornea fibrous structures remain largely intact. With additional increase in temperature, the corneal SHG intensity starts to increase and first reaches a peak value at 53°C before reaching a main peak at 65°C. Histological examination of the cornea at 53°C shows that although the collagen fibers remain visible, they have grouped into domains separated by cavities. This phenomenon becomes even more apparent as the temperature increased. Finally, at 90°C, the cavities are no longer visible and the cornea specimen seems to have merged into one continuous structure. These observations suggest that the SHG signal peaks observed at 53, 65, and 74 to 77°C correspond to the organization of collagen fibers into locally dense bundles made possible by the thermal disruption of cross-linking elements in the corneal stroma. The existence of multiple peaks suggests that the reorganization of corneal collagen occurs at three distinct phases between 53 and 77°C, corresponding to different strengths of cross-linking within corneal collagen. The absence of organized histological structure and the lack of SHG signal at 90°C suggest that at sufficiently high temperatures, neither the cross-linking nor the hydrogen bonds within corneal stroma has the strength to maintain the noncentrosymmetric structure of collagen fibrils responsible for generating the SHG signal.

It is interesting to compare our results with the differential scanning calorimetry (DSC) data from porcine corneas published previously. It was shown that the porcine cornea starts to undergo thermal denaturation at 54.5°C. At higher temperatures, porcine cornea absorbs more heat, reaching a maximum absorption temperature at 64.8°C. As the temperature further increases, the slope in the DSC thermogram changes until a baseline value is reached at 81.8°C. It was suggested that the slope changes in a DSC thermogram between 64.8 and 81.8°C correspond to the existence of subprocesses.¹⁷ Since we detected changes in SHG intensity starting at 30°C, while the DSC thermogram did not reveal corresponding changes in heat flow, it seems that SHG imaging is a more sensitive technique capable of detecting more subtle changes in corneal structures. In addition, since the same primary maximum transition temperature was measured using both techniques (SHG 65°C; DSC 64.8°C), SHG imaging is able to detect the structural change measured using DSC. Furthermore, the temperature range where DSC data indicated the existence of subprocesses also corresponds well to the SHG intensity diagram. Specifically, the SHG intensity plot demonstrated a variation in slope and the existence of a secondary SHG peak at 77°C. Despite the similarities of the SHG and DSC results, several differences do exist. First, the difference in baseline temperature (SHG 90°C; DSC 81.8°C) may be interpreted by the fact that we used intact porcine eyes for SHG imaging, while small excised corneal specimens were used in DSC measurements. The additional mechanical support provided by the sclera in maintaining the corneal structure in intact eyes may alter the response of thermally treated corneal collagen. Secondly, SHG imaging reveals a local maximum at 53°C. Such a change is missing in the DSC data, suggesting that the microscopic SHG imaging technique is more sensitive in revealing microscopic structural changes than the macroscopic heat flow measurement using DSC.

This work demonstrates that the nonlinear imaging modality of second harmonic generation microscopy may be used to obtain qualitative and quantitative information of collagen structural alterations in the cornea. Although SHG intensity is a far-field optical technique with spatial resolution in the micron range, the changes in SHG intensity are sensitive to alterations in cornea structures responsible for the second-order susceptibility tensor ${\chi }_2$ and other changes at the tissue level. In addition, since SHG microscopy depends on the intrinsic fiber orientations for signal generation, invasive histological or labeling procedures are not needed to obtain structural information valuable in guiding surgery and other therapeutic procedures of the cornea. Furthermore, since the SHG signal is a polarization effect rather than molecular excitation used in fluorescence microscopy, there is minimal photodamage to the specimens. Therefore, unlike other optical imaging techniques, SHG microscopy has the potential to be developed into an effective imaging technique for in vivo characterization of corneal structures. Our results show that SHG microscopy can be a valuable addition to the repertoire of imaging techniques in determining corneal structures, and may be developed into an effective imaging technology useful in guiding surgery and characterizing tissue engineered products intended for corneal repair.