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Journal of BIOPHOTONICS 134 B. R. Masters: Correlation of histology and linear and nonlinear microscopy of the living human cornea harmonic generation (SHG) an incident wave of frequency w generates a new signal at the frequency 2w. With the proper conditions the efficiency of this process can exceed 50 percent. SHG can only occur in media that does not contain inversion symmetry because all even-order nonlinear susceptibilities are zero in centrosymmetric media. SHG and third-harmonic generation (THG) can be explained by the theory of the nonlinear susceptibility in which for the time domain the polarization is expanded in a power series of the sum of products of the linear susceptibility and the electric field, the second-order susceptibility and the square of the electric field, and the third-order susceptibility and the cube of the electric field and higher-order terms. SHG is described by the second-order susceptibility, and THG is described by the third-order susceptibility [47]. The complete theoretical description of SHG and THG requires that the polarization and the vector properties of the electromagnetic field be accounted for in the analysis. THG has a broad potential for tissue imaging since it occurs in all materials, including dielectric materials with inversion symmetry [48]. In general THG is a weak process; however, it is dipole allowed. It occurs at all interfaces free from the constraint of phase-matching. Third-harmonic generation microscopy has a large potential for cell and tissue imaging. As new techniques are developed for the surface-enhanced THG at interfaces the technique may become useful for imaging the cornea. SHG microscopy in tissues can be used to image microtubles, oriented protein structures and stacked membranes [49–51]. Another emerging development is the use of THG microscopy with nano-gold particles to utilize the surface-plasmon-resonance effect [52]. Both SHG and THG imaging do not exhibit the saturation effects or the photobleaching effects that are associated with multiphoton excitation fluorescence microscopy. Therefore, SHG and THG microscopy do not require either intrinsic nor extrinsic fluorescent probes. These microscopic techniques can penetrate into millimeters of tissue and provide sub-micron three-dimensional optical sectioning. 3.3 Review of some key applications with nonlinear optical microscopy of the cornea In another investigation, the structure of the ex vivo rabbit cornea was studied with a multiphoton microscope that employed both SGH imaging and twophoton excited fluorescence (TPF) in a back scattering geometry [53]. Endogenous TPF and SHG signals from corneal cells and the extracellular matrix, re- spectively, were observed without exogenous dyes. The polarization dependence of the collagen SHG was used to study fiber orientation in the cornea. The authors demonstrated the spectra of the TPF fluorescence signal as well as the SHG signal in the collagen matrix of the cornea. The SHG signal had a quadratic dependence on the incident laser power. The authors compared the images from the TPF and the SHG modes with Hematoxylin and Eosin (H & E stain) stained en face formalin-fixed sections of the corneal stroma. Thus, they demonstrated the utility of correlative microscopy to interpret the images. Histology is fixed, stained tissue specimens is a useful comparison for optical imaging; however, the user should be cautious and knowledgeable about the artifacts of histology such as tissue shrinkage. Another study of corneal pathology in a transgenic mouse model shows the importance of measuring the emission spectra of the cornea to validate the interpretation of the nonlinear microscopy [54]. The authors used nonlinear optical microscopy (two photon excited fluorescence, and second harmonic generation signals) to image the corneas of a transgenic mouse model. Images were presented of the combined nonlinear signals from the cornea that consisted of TPF and SHG combined signals. To validate the images, H & E stained sections of the transgenic mouse cornea were presented. Metabolic activity in both the epithelial (the authors did not specify which cell layer of the epithelium was imaged) and the endothelial cellular layers were imaged by co-localizing the reduced pyridine nucleotide, NAD(P)H images and the flavin adenine dinucleotide (FAD) images in different colors. The authors also presented emission spectra for NAD(P)H and FAD in both the corneal epithelial and endothelial layers of the transgenic mouse. Unfortunately, the emission spectra were not corrected for the instrument response. Second-harmonic generation microscopy is another emerging area of nonlinear microscopy that is being applied to the cornea. SHG imaging of the excused porcine cornea after intrastromal femtosecond laser ablation was investigated to evaluate the next generation of laser surgical techniques [55]. This is an important approach since NIR fs laser surgery avoids the risks of mutagenicity and toxicity that accompanies the UV radiation of excimer lasers. Collagen has a noncentrosymmetric structure and can convert the incident ultrashort laser pulse to its second harmonic, thus providing a noninvasive imaging modality for the cornea. Since the SHG signal from the collagen in the cornea is emitted predominately in the forward direction (there was only a very weak signal in the back scattered path), it was detected in the transmission mode. The authors demonstrated that the SHG signal is proportional to the second power of the normalized illumination intensity. High # 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.biophotonics-journal.org
J. Biophoton. 2, No. 3 (2009) 135 contrast SHG images of corneal collagen fibers were obtained from the anterior corneal surface to about 200 micron depth within the cornea. The problem of forward and back scattered higherharmonic signals is the subject of another paper [56]. Collagen is the most abundant protein in the human body and exists in connective tissues including tendons, the skin, the cornea and the sclera. SHG imaging was used to image the porcine corneal collagen fibrils and to demonstrate their regular arrangement that accounts for corneal transparency. The sclera collagen fibrils are in a more inhomogeneous arrangement and that results in a lack of transparency. The SHG microscope was set up to acquire both the forward scattered SHG signal and also the backward scattered SHG signal. It should be stressed that only electron microscopy can image the collagen fibrils, since the diameters and the spacing of the individual fibrils are far below the diffraction limit of the microscope. The SHG emission field is a function of the size and the shape of the collagen fibrils, and is highly asymmetric due to the phase matching condition. It was previously demonstrated that objects with an axial size similar to the SHG wavelength shows forward directed SHG signals, while objects with an axial size less than l=10 produce equal SHG signals in both the forward and the backward directions [57]. Since electron microscopy demonstrates that the diameter of corneal collagen fibrils is approximately 30 nm, there should be significant backward SHG signal. Actual imaging showed the backward SHG signal from the cornea was extremely weak and there was no connection between the signals seen in the forward and the backward SHG imaging. In contrast to the cornea, the SHG images of the scleral collagen fibrils are similar in the forward and the backward scattering directions. The authors suggest that backward SHG imaging may provide a sensitive clinical method to study corneal haze or cloudiness. The next study shows how SHG microscopy is used to investigate collagen structure in the ex vivo human cornea. Second-harmonic generation imaging was used to identify differences in corneal stromal collagen between normal human and keratoconus corneas [58]. The authors examined six normal corneas from eye bank donors and 13 corneas of patients with keratoconus that were obtained after penetrating keratoplasty. A femtosecond titaniumsapphire laser with 800 nm output was used to generate second-harmonic signals at 400 nm from the central and the paracentral corneas. The authors found in keratoconus corneas there was less lamellar interweaving and a significant reduction or loss of lamellae inserting into Bowman’s layer. They conclude that compared with normal adult corneas, there were marked abnormalities in the structure of the anterior corneal collagen lamellae of keratoconus corneas. www.biophotonics-journal.org Several groups have demonstrated the efficacy of correlative microscopy in which multiphoton excitation microscopy and higher harmonic generation microscopy are used to image the same cornea [59, 60]. Multiphoton autofluorescence and second-harmonic generation images were obtained from whole ex vivo porcine eyes. These images were obtained from the cornea, the limbal region, the conjunctiva, and the sclera. The 780 nm output of a femtosecond, titanium-sapphire laser was used to induce autofluorescence in the region (435–700 nm) as well as secondgeneration signals at 390 nm. The SHG signals were used to image the collagen structures within the corneal stroma and the sclera. The authors obtained very weak SHG signals of the collagen, but they were able to determine that the collagen in the cornea is organized in a depth-dependent fashion, whereas the scleral collagen is more randomly packed. Other important applications of femtosecond lasers are in the area of using ultrashort laser pulses for refractive surgery. The use of ultrashort laser pulses (100–200 fs) can be used to generate microplasmas inside the cornea stromas [61, 62]. These plasmas can cut inside the tissue while leaving the anterior corneal layers intact. The authors found that the extent of thermal and mechanical damage to adjacent tissue is limited to 1 mm. Therefore, these lasers have a potential for use in intrastromal refractive surgery. 4. Discussion REVIEW ARTICLE The problems of interpretation of all types of microscopic images are formidable. There are optical aberrations in the imaging system and in the human eye, as well as artifacts due to specimen preparation of ex vivo specimens. One solution to the problem of aberrations is to incorporate an adaptive optics system (consisting of a wavefront sensor, a deformable mirror, and a feed-back control circuit) to minimize the spherical aberrations from the eye [61]. A second problem is how to minimize the error of interpretation of corneal images. It is suggested that the use of correlative microscopy, in which similar specimens are imaged with multiple microscopic techniques, is helpful. An exemplar of this technique is a study of the structure of the lens fibers in ex vivo human lenses containing cataracts. A correlation of both reflected light confocal microscopy on a freshly excised human lens and the subsequent fixing, embedding, and microtome sectioning for scanning electron microscopy of the identical regions of the ocular lens, confirmed the interpretation of the confocal images [63]. Another example is the comparison of confocal images from the in vivo images of the cornea with ex vivo corneal whole mounts that # 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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