In ophthalmology, a high-contrast, high-resolution, and large-area imaging technique with the potential of in vivo applications is of immense value in physiological studies and disease diagnosis. Although multiphoton imaging remains to be within the realm of surface tissue imaging, this approach can be readily applied to the ocular surface for obtaining images with subcellular resolution. To begin with, changes to the corneal structures are important in vision correction procedures. In laser in situ keratomileusis (LASIK) or conductive keratoplasty, the ability to monitor structural change of cornea collagen is vital in assessing the success of these procedures.
23 24 25 26 Although histologic procedures are available to monitor changes to the cornea, the transparent nature of the cornea prevents its internal structures to be analyzed easily.
27 Although x-ray diffraction techniques have added valuable information to the organization of collagen fibers in the cornea,
28 29 30 31 a direct visualization technique to obtain structural information about the cornea in intact eyes would be of immense value in clinical evaluation of corneal states, and multiphoton collagen imaging offers such possibilities.
20 21 22 In addition, the limbus, the junctional structure separating the transparent cornea and opaque sclera, is important in maintaining corneal epithelial tissues. Structurally, the limbus contains stem cells that differentiate into corneal epithelium. For ophthalmologists, the ability to monitor the physiological states within the limbus without histologic fixation procedures has potential application in tissue engineering.
32 33 Specifically, if multiphoton microscopy can be used to image the limbal epithelium, the spectral separation of cellular autofluorescence from the SHG signal of the surrounding cornea collagen fibers will enable high-contrast imaging of the limbus to be achieved. Finally, because the conjunctiva and sclera are respectively organized into connective tissues of the outer edges of the eye, one can anticipate multiphoton autofluorescence and SHG imaging to be useful in spectrally resolving the structures of the sclera from the cornea. In vivo imaging of the sclera is invaluable in assessing tissue states in clinical settings. For example, thermal treatment of the sclera has been used for treatment of ocular melanoma.
34 However, the ideal scenario would be the eradication of the tumor mass without damaging the surrounding connective tissues. Because the SHG signal is sensitive to the thermal denaturation of collagen fibers, a potential application of SHG imaging is to assess the degree of thermal damage to the sclera without histologic procedures.
35 Finally, the change of autofluorescence in diabetic patients and diseases such as corneal and conjunctival tumors are potential imaging targets of multiphoton microscopy.
36 37 38