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Confocal and Multiphoton Imaging of Cornea
Confocal and Multiphoton Imaging of Cornea Gopal S Jayabalan and Josef F Bille, University of Heidelberg, Heidelberg, Germany r 2018 Elsevier Ltd. All rights reserved.
Introduction Most of our daily activities depend on a proper vision, and any loss in the visual acuity will strongly influence our quality of life. Preserving vision throughout an individual’s lifetime is of a major concern in ophthalmology. The reliable diagnoses in ophthalmology are determined by detecting and monitoring the infinitesimal changes in tissue structures. The ophthalmologists mostly rely on slit-lamp biomicroscopy that was developed over a century ago in the 1890s. Subjective slit-lamp biomicroscopy remains as the backbone of clinical examination; however, objective tools and technologies are needed in order to provide a comprehensive understanding of structural alterations at a cellular level. Corneal disease is one of the leading causes of blindness worldwide, and a prompt and accurate diagnosis of corneal diseases is a milestone for successful treatment and followup. Since the invention of the first ophthalmoscope by Hermann von Helmholtz in 1851, the quest for quantitative in vivo imaging of ocular surfaces for diagnostic and monitoring purposes was initiated. Recently, a number of imaging technologies were developed for research and clinical purposes. Boosted by the newest developments in microscopy, confocal microscopy has been widely employed in clinics and research for corneal diagnosis. Confocal microscopy has emerged as a powerful technique to study the corneal structure (Fig. 1) with high spatial and contrast images compared to conventional microscopy. With the introduction of ultrafast pulsed laser sources, multiphoton microscopy practically became feasible and experienced a tremendous success in biological microscopy. Multiphoton microscopy allows three-dimensional (3D) tissue imaging over time, and it is an important asset in investigational imaging. Intrinsic tissue fluorescence, called autofluorescence, which is initiated by collagen, elastin, NAD(P)H etc., can be imaged by multiphoton microscopy and provides structural and functional information of the cornea. In this article, we will discuss the principles and the potential use and limitations of confocal and multiphoton microscopy for corneal imaging.
Principles of Confocal and Multiphoton Microscopy Confocal Microscopy The principle of confocal microscopy was patented in 1957 by Marvin Minsky, in order to overcome the limitations of conventional wide-field microscopy. In a conventional wide-field microscope, the entire specimen (i.e., cornea) will be illuminated evenly by a light source, and the specimens in the optical path are all excited at the same time. The resulting fluorescence (i.e., emitted light) from the specimen will be detected by the photodetector or camera including a largely unfocused background part. In contrast, confocal microscopy uses a point illumination source with a pinhole in front of the detector to eliminate the out of
Fig. 1 Anatomical structure of cornea: (a) schematic presentation of a cornea (http://www.allaboutvision.com/visionsurgery/corneal-inlays-onlays. htm) and (b) layers of cornea (courtesy from the slide Krstic, R.V., 1991. Human microscopic anatomy. Springer).
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focus light or glare. The optical resolution of confocal microscopy images is better than conventional wide-field microscopy, since the fluorescence is produced very close to the focal plane.
Multiphoton Microscopy Multiphoton microscopy includes two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and third harmonic generation (THG) imaging (Fig. 2). In 1931, the theoretical basis of two-photon excitation was established by Maria Göppert-Mayer, and then the photophysical effect of two-photon excitation was experimentally verified by Kaiser and Garret in 1963. Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by simultaneous absorption of two photons. The familiar single-photon (i.e., confocal) fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This single-photon process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process occurs if the sum of energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Since this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance. Under sufficient intense excitation, three-photon and higher photon excitation are also possible. This process is similar to two-photon excitation wherein three photons or higher number of photons must interact with the fluorophore simultaneously to produce the fluorescence. Almost all multiphoton microscopy is based on ultrashort pulsed lasers, because the multiphoton efficiency of an n-photon process depends on the peak power P according to Pn relation. The use of pulsed lasers enables efficient fluorophore excitation at a fast scanning rate. Moreover, focusing a pulsed laser into a specimen can generate a harmonic upconversion as well. Although the nonlinear optical effect known as SHG has been recognized in early days of laser physics, the use of SHG and its three-photon variant third (THG) is fairly new in research. Harmonic generation is a nonlinear process in which photons with the same frequency interact with a nonlinear material and generate a new photon with twice the energy. SHG is useful for investigating the ordered structural protein assemblies such as collagen fibers in the cornea.
Confocal Laser Scanning Microscopy Confocal laser scanning microscopy (CLSM) has been widely implemented in clinics for diagnostic purposes. It is based on a conventional optical microscope, instead of a lamp a laser is used as a light source. The laser is focused on the cornea through the objective lens to a single spot in the focal plane. The laser beam is scanned in a raster pattern using the scanning mirrors in x–y direction and an entire image of the cornea at a certain focal plane will be reconstructed. The emitted light generated from the cornea at the focal plane will be collected by the same objective lens and the collected signals will be recorded using a photomultiplier tube (PMT). This scanning principle does not improve the spatial resolution over conventional optical microscopy, because the excitation is produced throughout the entire beam path of the laser in the cornea, i.e., above and below the focal plane. However, the spatial resolution can be improved by placing a pinhole in front of the detector to eliminate the out of focus light. This leads to an excellent spatial resolution and enables serial optical sections from thick specimens (Fig. 3(a)).
Fig. 2 Jablonski diagram of single-photon excitation, two-photon excitation (2PE), second harmonic generation (SHG), and third harmonic generation (THG). (http://www.azooptics.com/Article.aspx?ArticleID=1175)
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Fig. 3 The principle of confocal and two-photon microscopy. (a) In confocal microscopy, the emitted fluorescent light from the focal plane will pass through the pinhole and the out of focus light will be blocked by the pinhole and (b) in multiphoton microscopy, the two-photon excitation generates fluorescence only at the focal plane, so a pinhole is not required since no background fluorescence is produced. Reproduced from Allocca, G., Kusumbe, A.P., Ramasamy, S.K., Wang, N., 2016. Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models. BoneKEy Reports 5. doi:10.1038/bonekey.2016.84.
Two-Photon Laser Scanning Microscopy The invention of two-photon fluorescence light microscopy by Denk, Webb, and coworkers revolutionized the in vivo 3D cellular imaging. By focusing an ultrashort pulsed laser through the objective lens sufficient excitation intensity can be generated on the focal plane (Fig. 3(b)). When the laser beam is focused on the specimen, the photons become crowded and the probability of two photons interacting simultaneously will be increased. The significant advantage of two-photon excitation over single-photon (confocal) excitation is the narrow localization. In confocal microscopy, although the fluorescence is excited throughout the illuminated volume of the specimen, the signal originating from the focal plane will pass through the pinhole. In two-photon excitation, the fluorescence will be generated only at the focal plane, and no background fluorescence will be produced. Therefore, a pinhole is not required for two-photon microscopy. Due to the localized excitation of two-photon excitation, the 3D resolution of the images can be achieved identically to confocal microscopy. Since there is no absorption in out of focus specimen area, the excitation light can penetrate much deeper through the specimen to the focal plane. This results in greater penetration compared to confocal microscopy. Also, two-photon microscopy minimizes photobleaching and photodamage.
Confocal and Multiphoton Microscopy of Cornea Three types of confocal microscopes are available for corneal imaging:
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Tandem scanning microscopy. Slit scanning microscopy. Laser scanning microscopy.
Tandem scanning microscopy sheds the light of high intensity through a pinhole and generates a two-dimensional (2D) image. However, tandem scanning microscopy for confocal imaging is not produced anymore. In contrast to tandem scanning, slit scanning microscopy has been employed in clinics with reduced scanning and examination time with high contrast imaging. Slit scanning has the ability to scan many points in parallel to the axis of the slit. Laser scanning microscopy is the latest addition to corneal imaging, where the laser beam is scanned over the back of the microscope objective through scanning mirrors. The Heidelberg Retina Tomograph (HRT) in combination with the Rostock Cornea Module (RCM) is a commercially available
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Fig. 4 (a) Heidelberg Engineering Heidelberg retinal tomograph-rostock cornea module (HRT-RCM) confocal microscopy and (b) schematic diagram of the optical set up of the HRT-RCM. Heidelberg Engineering Brochure.
confocal microscope that has been widely used in clinics for corneal imaging. The HRT-RCM uses a class I laser and this laser poses no risk of ocular injury.
Heidelberg Retinal Tomograph-RCM The Heidelberg Retinal Tomograph-RCM (HRT-RCM) (Fig. 4(a)) was developed by Heidelberg Engineering GmbH in cooperation with Rostock Eye clinic for in vivo investigation of the cornea. A 670-nm laser is used as light source and the laser is focused on the cornea with a high numerical aperture objective lens (63 /0.95 NA or 60 /0.90 NA; interchangeable). The laser is raster scanned by the scanning mirrors and is focused on the cornea by the objective lens. The emitted fluorescent light from the cornea is captured using a detector. The confocal pinhole is used in front of the detector to eliminate the out of focus light (Fig. 4(b)). The confocal imaging of the cornea can be acquired with the field of view 250 mm 250 mm, 400 mm 400 mm, and 500 mm 500 mm with a 63 objective lens. A viscous sterile gel is interposed between the objective lens and cornea to eliminate the optical interface between two different refractive indices. The depth of the focus can be adjusted manually relative to the cornea in a plane parallel to the corneal surface, to scan the cornea from epithelium to endothelium.
Confocal Imaging of Normal Cornea The HRT-RCM provides excellent resolution with sufficient contrast at different layers of the cornea, and distinct corneal layers from epithelium to endothelium can be visualized. The corneal epithelium is composed of five to six layers (Fig. 5(a)–(d)), as superficial cells, intermediate cells, and basal cells. The superficial cells are flat polygonal cells with well-defined border, prominent nuclei and uniform density of cytoplasm. It is about 40–50 mm in diameter and approximately 5 mm thick. The intermediate cells also known as wing cells are similar to superficial cells, but the nuclei are not evident. It is typically about 30–45 mm in size and characterized by the cell borders. Basal cells are approximately 10–15 mm and smaller in size. The basal cells appear denser than the superficial and wing cells. The subbasal nerve plexus (Fig. 5(e)) is characterized by the presence of hyper-reflective fibers of 4–8 mm length, and connected with anastomoses and organized in a vortex pattern in the lower nasal quadrant of the paracentral cornea. The visualization of corneal nerve plexus was not possible until the introduction of confocal microscopy. The nerve fibers appear bright and well contrasted against a dark background. Corneal stroma accounts for 90% of total corneal thickness and the keratocytes density is much higher in anterior stroma than in posterior stroma. The keratocyte counts are 1000 cells/mm2 in the anterior stroma and 700 cells/mm2 in the posterior stroma. The confocal image of the stroma exhibits multiple irregular oval, round or bean shaped bright structures representing keratocytes (Fig. 5(f) and (g)). The collagen fibers cannot be visualized by confocal microscopy in normal cornea. Corneal endothelium is composed of a single layer of cells that are 4–6 mm thick and approximately 20 mm in diameter. The endothelial cells progressively decline with age, and the normal endothelial cell count amounts to 3000 cells/mm2 in a healthy cornea. The endothelial cells appear as bright hexagonal and polygonal cells with unrecognizable nuclei (Fig. 5(h)).
Confocal Microscopy in Corneal Pathologies Confocal microscopy has been an effective tool in clinical diagnosis, and it is widely used to assess corneal infections, corneal dystrophies, pre and postsurgical assessment of laser-assisted in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), keratoconus and infectious keratitis. Also, it is used to monitor the cornea after injuries, and for differential diagnosis of
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Fig. 5 In vivo confocal microscopy images of the corneal layers in normal human subject from epithelium to endothelium. (a)–(d) Epithelium, (e) subbasal nerve plexus, (f) and (g) anterior and posterior stroma, and (h) endothelium. Courtesy of Prof. R. Guthoff, Prof. J. Stave, Dr. A. Zhivov, University of Rostock (from Heidelberg PowerPoint).
Fig. 6 In vivo confocal microscopy images demonstrating different forms of Langerhans cells. (a) Individual cell bodies without processes; (b) cells bearing dendrites; and (c) cells arranged in a meshwork via long interdigitating dendrites. Reproduced from Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Ophthalmology. Springer: Germany.
conjunctival tumors and conjunctivitis. As an example for clinical confocal imaging of the cornea, the Langerhans cell distribution and postsurgical assessment of LASIK using the HRT-RCM is shown in Figs. 6 and 7.
Langerhans Cells Langerhans cells can be evaluated using confocal microscopy with emphasis on cell morphology and cell distribution. In confocal microscopy, the Langerhans cells are visualized typically as bright corpuscular particles with dendritic morphology. These cells are distributed in low numbers at the center of the cornea and higher densities at the periphery of the cornea. Confocal microscopy helps in differentiating the Langerhans cells bodies that lack dendrites, Langerhans cells with small dendritic process form a local network, and Langerhans cells forming a meshwork via long interdigitating dendrites (Fig. 6(a)–(c)). The average density of Langerhans cells in normal subjects is 3473 cells/mm2 (range 0–64 cells/mm2) in the center of the cornea, and 9878 cells/mm2 (range 0–208 cells/mm2) in the periphery. Langerhans cells contribute to the immune and inflammatory responses, and determine the cell-mediated immunity.
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Fig. 7 Innervation after refractive surgery. (a) Nerve fibers near subbasal layer; (b) nerve fibers near Bowman’s membrane; (c) two months after LASIK – small regenerating nerve fibers in the central cornea; and (d) nine months after LASIK – new nerve fiber growing from the edge of flap region to center. Reproduced from Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Ophthalmology. Springer: Germany.
Postsurgical Assessment of LASIK LASIK is an excimer laser-based refractive surgical procedure that is currently being successfully used by surgeons to correct refractive errors (myopia, hyperopia etc.). After LASIK surgery, the cornea is typically evaluated by slit-lamp biomicroscopy and computerized corneal topography. The confocal microscopy evaluation of LASIK pre- and postsurgery adds new aspects in corneal diagnosis. The distribution of the corneal nerves and degeneration of the nerve structures can be visualized by confocal microscopy, and thus a direct comparison between innervation and sensitivity or symptom severity of the dry eye can be provided (Fig. 7).
Two-Photon Laser Scanning Microscopy Two-photon laser scanning microscopy based on the HRT was developed to investigate the cornea (Fig. 8). A compact femtosecond laser was employed in the system as the light source and an additional detection path for the two-photon mode was implemented. Both the confocal and two-photon images can be captured using the same instrument. An electronic switch facilitates the switching between the two imaging modes, while both detection modes are synchronized with the scanning unit. The laser beam is deflected in x- and y-direction via two scanning mirrors (scan unit, Fig. 8(b)) to trace out a square raster of 300 300 mm², 200 200 mm², or 150 150 mm² at a frame rate up to 16 Hz. The laser is focused on the cornea with a high numerical aperture objective lens (40 /0.80 NA or 16 /0.80 NA; interchangeable). The wide field of view can be achieved by using the 16 objective lens with less distinct cell morphology. Like confocal microscopy (HRT-RCM) the focus can be adjusted manually and different layers of the cornea can be visualized. A viscous sterile gel has to be placed between the objective lens and the cornea to eliminate the optical interface between two different indices.
Two-Photon Imaging of Normal Cornea The TPEF imaging of cornea has been extensively employed in preclinical studies, but has not been implemented in clinics yet. Although confocal microscopy has been used in clinical diagnosis, the collagen fiber orientation in the stroma cannot be visualized, and it provides only morphological information rather than the tissue’s functional state. With two-photon imaging
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Fig. 8 (a) Heidelberg Engineering two-photon microscopy and (b) schematic diagram of the optical set up of two-photon microscopy based on HRT. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Fig. 9 Two-photon imaging of corneal epithelial cells (a) and stromal collagen fibers (b) of human cornea. Reproduced from Qu, Y., Thomas, K. E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
both the tissue structures and the metabolic state can be well addressed. Two-photon microscopy provides excellent resolution and high contrast images of the cornea similar to confocal microscopy. High resolution two-photon images of the corneal epithelium and the collagen fibers from the human corneal buttons are shown in Fig. 9. The collagen fibers in the stroma can be well established with TPEF and SHG imaging.
Two-Photon Imaging of Limbal Stem Cells The limbal stem cells are located in the basal layer of the corneal limbus, and the proliferation of the limbal cells maintains the cornea by replacing the lost cells by tears. Limbal stem cells also prevent the conjunctival epithelial cells from migrating onto the surface of the cornea. The limbal stem cells have never been visualized with confocal microscopy and with the use of two-photon microscopy the limbal stem cells have been identified in mice. The limbal stem cells are smaller in size and it is shown by twophoton microscopy (Fig. 10). The two-photon imaging of limbal stem cells would help in the diagnosis of limbal stem cell deficiency and also differentiate and explore the nature of the limbal stem cells in a precise way.
Two-Photon Imaging of Infectious Keratitis Confocal microscopy plays a significant role in detecting the microorganisms causing corneal infections. It is difficult to identify the microorganisms like Acanthamoeba on the ocular surfaces since the appearance can impersonate as herpetic or bacterial keratitis. In most cases, the Acanthamoeba keratitis is diagnosed either by tissue culture or corneal biopsy, and histological analysis.
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Fig. 10 Two-photon imaging of mice limbal stem cells. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Fig. 11 Two-photon imaging of Acanthamoeba keratitis. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Although confocal microscopy lacks sufficient resolution in detecting the Acanthamoeba keratitis, it is used for diagnostic purposes since confocal microscopy is the only available diagnostic tool for patients. In confocal microscopy, the Acanthamoeba is visualized as round or ovoid highly reflective structures and the same is depicted by the two-photon microscopy (Fig. 11).
Second Harmonic Imaging of Cornea Collagen is the major component of connective tissues including cornea, and due to its transparency to visual and near infrared light the human eye is ideal for laser-based diagnostic and imaging applications. Among different multiphoton implementations, SHG imaging is particularly suitable to investigate non-centrosymmetric structures like collagen fibrils (Figs. 12 and 13). The basic structure of the collagen fibril is a triple helix composed of three proteins chains, which gives collagen the intrinsic ability of SHG
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Fig. 12 SHG imaging of corneal collagen fibrils in (a) forward and (b) backward directions from porcine cornea. The fibrillar structures resolved in (a) correspond to collagen bundles which are composed of regularly packed collagen fibrils. Scale¼10 mm. Reproduced from Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797.
Fig. 13 SHG imaging and two-photon imaging of human cornea from (a) Bowman’s membrane and (b) corneal stroma. The collagen fibers were visualized using the SHG imaging and the keratocytes (arrows) were visualized with the two-photon imaging. Reproduced from Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797.
imaging. Similar to TPEF, SHG is produced in only a small focal volume, permitting high resolution 3D optical sectioning of thick tissues. In contrast to TPEF, SHG from collagen is an intrinsic and a coherent process. Intrinsic imaging avoids the complications of slicing and labeling, and samples can be investigated under physiological conditions. Coherent constructive or destructive interference of SHG provides extra hints to the ultrastructure of collagen fibrils and their organizations. The collagen fibrils of the porcine cornea using SHG imaging are shown in Fig. 12. Most collagen fibrils run parallel to the cornea surface and share similar orientations with their neighbors. One of the unique features of SHG is that the geometry of the SHG emission field reflects the size and shape of the collagen fibrils. The SHG radiation pattern is mainly determined by the phase matching condition and the objects with the axial size on the order of the second harmonic wavelength exhibit forward directed SHG, while objects with a axial size less than l/10 (approx. 40 nm) are estimated to produce nearly equal backward and forward SHG signals. In Fig. 12(b), the backward SHG from cornea was extremely weak and there was no correlation between the structures revealed by forward and backward SHG imaging. The predominant forward SHG implies that the corneal collagen fibrils are not randomly distributed. It’s worth mentioning that the fibrillar structures revealed in Fig. 12(b) actually correspond to collagen bundles composed of many corneal collagen fibrils. In cornea, the collage fibrils (n ¼1.47) and the extrafibrillar matrix (n ¼ 1.35) have different refractive indices, thus the collagen fibrils have to be treated as individual scatters. Since the dimension of the collagen bundle (approximately 0.5 mm) is close to the second harmonic wavelength (400 nm), the phase correlation of SHG from neighboring collagen fibrils should be taken into account. The forward predominant SHG radiation from cornea indicates the regular arrangement of the collagen fibrils, at least in the domain of the collagen bundles. In Fig. 13, SHG and two-photon imaging of human cornea from Bowman’s membrane (Fig. 13(a)) and corneal stroma (Fig. 13 (b)) is depicted. The collagen fibers were visualized using the SHG imaging and the keratocytes (arrows) were imaged with TPEF imaging.
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Fig. 14 Two-photon microscopy of enucleated rabbit eyes 2 weeks after standard corneal CXL, upper row: two-photon imaging at 710 nm excitation light and lower row: second harmonic generation. (a) Two-photon imaging of the central zone of control cornea; (b) the peripheral zone of the treated cornea shows keratocytes in a dense network; (c) at the tangential zone, there is a sharp transition between the treated area and the untreated area, as shown by diminished keratocytes and a strong autofluorescence signal toward the central zone; (d) in the central zone, there are no keratocytes and the autofluorescence signal is uniform and strong; (e) and (f) second harmonic generation of stromal collagen in the control zone and the peripheral zone of the treated cornea shows a wavelike pattern; (g) at the transition zone toward the treated area, the wavelike pattern changes to a uniform signal; (h) in the central zone, a uniform SHG signal is visible. Reproduced from Steven, P., Hovakimyan, M., Guthoff, R.F., Hüttmann, G., Stachs, O., 2010. Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements. Journal of Cataract Refractive Surgery 36 (12), 2150–2159.
Two-Photon Imaging of Corneal Cross-linking Keratoconus is a progressive, noninflammatory and bilateral ectatic corneal disease characterized by corneal thinning and weakening. The thinning is most apparent at the apex of the cornea, and the steep conical protrusion leads to high myopia with irregular astigmatism. The visual loss occurs primarily from irregular astigmatism and myopia, and secondarily from the corneal scarring. Computerized corneal topography has become a gold standard for the early identification of keratoconus and recently confocal microscopy has also been used to assess the keratoconus cornea. Corneal cross-linking (CXL) is an invasive treatment procedure for keratoconus in order to stop or delay the process. The CXL procedure increases the mechanical and biomechanical stability of the cornea by application of ultraviolet-A light of 370 nm and riboflavin (vitamin B2). The assessment of cornea after cross-linking is limited because the treatment effect cannot be visualized by slit-lamp biomicroscopy. Confocal microscopy can detect the side effects like stromal edema and rarefication, the reappearance of stromal keratocytes over time. However, the effect of crosslinks between the collagen fibers after CXL cannot be visualized by confocal microscopy. Two-photon imaging with SHG is effective in measuring the crosslinks between collagen fibers as shown in Fig. 14.
Laser Safety Considerations of Two-Photon Microscopy The safe use of lasers has been a primary consideration since lasers are widely used for treatments and in research laboratories. The American National Standards Institute (ANSI) has specific standards for eye safety that take into account many of the important experimental variables such as laser power delivered at the cornea, viewing time, beam diameter or full-field viewing, the exposure area on the retina, and the wavelength. Near infrared radiation (NIR) relative to visible wavelength is considered to be less
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phototoxic and has deeper penetration in thick biological tissues, which is one of the key reasons for its widespread use in imaging devices. Compared to retinal imaging, the safety standards for illuminating the cornea and anterior structures of the eye are tolerant. The laser safety limits for corneal imaging can be calculated by the maximum permissible exposures (MPE) according to the ANSI standards as follows: Recommended MP corneal irradiance ¼ 25 t 0:75 W=cm2 for to10s Recommended MP corneal irradiance ¼ 4:0 W=cm2 for t410s For example, if the irradiated area is described by 1536 mm 1536 mm¼0.0236 cm2, the MPE should be 94.4 mW for longer exposure time (t410s) and should be 515 mW for shorter exposure time.
Future Directions Before implementing multiphoton microscopy for in vivo imaging of the human cornea, the shortcomings of these nonlinear microscopy techniques have to be addressed. For TPEF imaging, the drawback of depositing the energy into the tissue has to be evaluated to prevent photodamage. Although SHG and THG are absorption-free and can be used without photobleaching effects, harmonic imaging has another disadvantage that currently obstructs its use in ophthalmic practice. The harmonic signals are emitted predominantly in forward direction, whereas in backward direction the harmonic signals are weak. This limits SHG imaging for in vivo applications, and the backscattered signal might be enhanced using an absorbing dye. Also, new optical techniques such as wavefront aberration optimization and adaptive optics may be a key for future in vivo applications. Until these restrictions of multiphoton microscopy are eliminated, confocal microscopy remains exclusive in the assessment of cellular morphology in living human cornea.
Acknowledgment The authors like to thank Heidelberg Engineering GmbH (Heidelberg, Germany) for providing equipment support.
See also: Optical Coherence Tomography and Its Application to Imaging of Skin and Retina. Optics of the Human Eye
Further Reading Denk, W., Strickler, J.H., Webb, W.W., 1990. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76. Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Opthalmology. Germany: Springer. Guthoff, R.F., Zhivov, A., Stachs, O., 2009. In vivo confocal microscopy, an inner vision of the cornea – A major review. Clinical and Experimental Ophthalmology 37, 100–117. Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797. Hao, M., Flynn, K., Nien-Shy, C., et al., 2010. In vivo non linear optical (NLO) imaging in live rabbit eyes using the heidelberg two-photon laser ophthalmoscope. Experimental Eye Research 91 (2), 308–314. Mantopoulos, D., Cruzat, A., Hamrah, P., 2010. In vivo imaging of corneal inflammation: New tools for clinical practice and research. Seminars in Ophthalmology 25 (5-6), 178–185. Steven, P., Hovakimyan, M., Guthoff, R.F., Hüttmann, G., Stachs, O., 2010. Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements. Journal of Cataract & Refractive Surgery 36 (12), 2150–2159.
Introduction Most of our daily activities depend on a proper vision, and any loss in the visual acuity will strongly influence our quality of life. Preserving vision throughout an individual’s lifetime is of a major concern in ophthalmology. The reliable diagnoses in ophthalmology are determined by detecting and monitoring the infinitesimal changes in tissue structures. The ophthalmologists mostly rely on slit-lamp biomicroscopy that was developed over a century ago in the 1890s. Subjective slit-lamp biomicroscopy remains as the backbone of clinical examination; however, objective tools and technologies are needed in order to provide a comprehensive understanding of structural alterations at a cellular level. Corneal disease is one of the leading causes of blindness worldwide, and a prompt and accurate diagnosis of corneal diseases is a milestone for successful treatment and followup. Since the invention of the first ophthalmoscope by Hermann von Helmholtz in 1851, the quest for quantitative in vivo imaging of ocular surfaces for diagnostic and monitoring purposes was initiated. Recently, a number of imaging technologies were developed for research and clinical purposes. Boosted by the newest developments in microscopy, confocal microscopy has been widely employed in clinics and research for corneal diagnosis. Confocal microscopy has emerged as a powerful technique to study the corneal structure (Fig. 1) with high spatial and contrast images compared to conventional microscopy. With the introduction of ultrafast pulsed laser sources, multiphoton microscopy practically became feasible and experienced a tremendous success in biological microscopy. Multiphoton microscopy allows three-dimensional (3D) tissue imaging over time, and it is an important asset in investigational imaging. Intrinsic tissue fluorescence, called autofluorescence, which is initiated by collagen, elastin, NAD(P)H etc., can be imaged by multiphoton microscopy and provides structural and functional information of the cornea. In this article, we will discuss the principles and the potential use and limitations of confocal and multiphoton microscopy for corneal imaging.
Principles of Confocal and Multiphoton Microscopy Confocal Microscopy The principle of confocal microscopy was patented in 1957 by Marvin Minsky, in order to overcome the limitations of conventional wide-field microscopy. In a conventional wide-field microscope, the entire specimen (i.e., cornea) will be illuminated evenly by a light source, and the specimens in the optical path are all excited at the same time. The resulting fluorescence (i.e., emitted light) from the specimen will be detected by the photodetector or camera including a largely unfocused background part. In contrast, confocal microscopy uses a point illumination source with a pinhole in front of the detector to eliminate the out of
Fig. 1 Anatomical structure of cornea: (a) schematic presentation of a cornea (http://www.allaboutvision.com/visionsurgery/corneal-inlays-onlays. htm) and (b) layers of cornea (courtesy from the slide Krstic, R.V., 1991. Human microscopic anatomy. Springer).
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focus light or glare. The optical resolution of confocal microscopy images is better than conventional wide-field microscopy, since the fluorescence is produced very close to the focal plane.
Multiphoton Microscopy Multiphoton microscopy includes two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and third harmonic generation (THG) imaging (Fig. 2). In 1931, the theoretical basis of two-photon excitation was established by Maria Göppert-Mayer, and then the photophysical effect of two-photon excitation was experimentally verified by Kaiser and Garret in 1963. Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by simultaneous absorption of two photons. The familiar single-photon (i.e., confocal) fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This single-photon process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process occurs if the sum of energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Since this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance. Under sufficient intense excitation, three-photon and higher photon excitation are also possible. This process is similar to two-photon excitation wherein three photons or higher number of photons must interact with the fluorophore simultaneously to produce the fluorescence. Almost all multiphoton microscopy is based on ultrashort pulsed lasers, because the multiphoton efficiency of an n-photon process depends on the peak power P according to Pn relation. The use of pulsed lasers enables efficient fluorophore excitation at a fast scanning rate. Moreover, focusing a pulsed laser into a specimen can generate a harmonic upconversion as well. Although the nonlinear optical effect known as SHG has been recognized in early days of laser physics, the use of SHG and its three-photon variant third (THG) is fairly new in research. Harmonic generation is a nonlinear process in which photons with the same frequency interact with a nonlinear material and generate a new photon with twice the energy. SHG is useful for investigating the ordered structural protein assemblies such as collagen fibers in the cornea.
Confocal Laser Scanning Microscopy Confocal laser scanning microscopy (CLSM) has been widely implemented in clinics for diagnostic purposes. It is based on a conventional optical microscope, instead of a lamp a laser is used as a light source. The laser is focused on the cornea through the objective lens to a single spot in the focal plane. The laser beam is scanned in a raster pattern using the scanning mirrors in x–y direction and an entire image of the cornea at a certain focal plane will be reconstructed. The emitted light generated from the cornea at the focal plane will be collected by the same objective lens and the collected signals will be recorded using a photomultiplier tube (PMT). This scanning principle does not improve the spatial resolution over conventional optical microscopy, because the excitation is produced throughout the entire beam path of the laser in the cornea, i.e., above and below the focal plane. However, the spatial resolution can be improved by placing a pinhole in front of the detector to eliminate the out of focus light. This leads to an excellent spatial resolution and enables serial optical sections from thick specimens (Fig. 3(a)).
Fig. 2 Jablonski diagram of single-photon excitation, two-photon excitation (2PE), second harmonic generation (SHG), and third harmonic generation (THG). (http://www.azooptics.com/Article.aspx?ArticleID=1175)
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Fig. 3 The principle of confocal and two-photon microscopy. (a) In confocal microscopy, the emitted fluorescent light from the focal plane will pass through the pinhole and the out of focus light will be blocked by the pinhole and (b) in multiphoton microscopy, the two-photon excitation generates fluorescence only at the focal plane, so a pinhole is not required since no background fluorescence is produced. Reproduced from Allocca, G., Kusumbe, A.P., Ramasamy, S.K., Wang, N., 2016. Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models. BoneKEy Reports 5. doi:10.1038/bonekey.2016.84.
Two-Photon Laser Scanning Microscopy The invention of two-photon fluorescence light microscopy by Denk, Webb, and coworkers revolutionized the in vivo 3D cellular imaging. By focusing an ultrashort pulsed laser through the objective lens sufficient excitation intensity can be generated on the focal plane (Fig. 3(b)). When the laser beam is focused on the specimen, the photons become crowded and the probability of two photons interacting simultaneously will be increased. The significant advantage of two-photon excitation over single-photon (confocal) excitation is the narrow localization. In confocal microscopy, although the fluorescence is excited throughout the illuminated volume of the specimen, the signal originating from the focal plane will pass through the pinhole. In two-photon excitation, the fluorescence will be generated only at the focal plane, and no background fluorescence will be produced. Therefore, a pinhole is not required for two-photon microscopy. Due to the localized excitation of two-photon excitation, the 3D resolution of the images can be achieved identically to confocal microscopy. Since there is no absorption in out of focus specimen area, the excitation light can penetrate much deeper through the specimen to the focal plane. This results in greater penetration compared to confocal microscopy. Also, two-photon microscopy minimizes photobleaching and photodamage.
Confocal and Multiphoton Microscopy of Cornea Three types of confocal microscopes are available for corneal imaging:
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Tandem scanning microscopy. Slit scanning microscopy. Laser scanning microscopy.
Tandem scanning microscopy sheds the light of high intensity through a pinhole and generates a two-dimensional (2D) image. However, tandem scanning microscopy for confocal imaging is not produced anymore. In contrast to tandem scanning, slit scanning microscopy has been employed in clinics with reduced scanning and examination time with high contrast imaging. Slit scanning has the ability to scan many points in parallel to the axis of the slit. Laser scanning microscopy is the latest addition to corneal imaging, where the laser beam is scanned over the back of the microscope objective through scanning mirrors. The Heidelberg Retina Tomograph (HRT) in combination with the Rostock Cornea Module (RCM) is a commercially available
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Fig. 4 (a) Heidelberg Engineering Heidelberg retinal tomograph-rostock cornea module (HRT-RCM) confocal microscopy and (b) schematic diagram of the optical set up of the HRT-RCM. Heidelberg Engineering Brochure.
confocal microscope that has been widely used in clinics for corneal imaging. The HRT-RCM uses a class I laser and this laser poses no risk of ocular injury.
Heidelberg Retinal Tomograph-RCM The Heidelberg Retinal Tomograph-RCM (HRT-RCM) (Fig. 4(a)) was developed by Heidelberg Engineering GmbH in cooperation with Rostock Eye clinic for in vivo investigation of the cornea. A 670-nm laser is used as light source and the laser is focused on the cornea with a high numerical aperture objective lens (63 /0.95 NA or 60 /0.90 NA; interchangeable). The laser is raster scanned by the scanning mirrors and is focused on the cornea by the objective lens. The emitted fluorescent light from the cornea is captured using a detector. The confocal pinhole is used in front of the detector to eliminate the out of focus light (Fig. 4(b)). The confocal imaging of the cornea can be acquired with the field of view 250 mm 250 mm, 400 mm 400 mm, and 500 mm 500 mm with a 63 objective lens. A viscous sterile gel is interposed between the objective lens and cornea to eliminate the optical interface between two different refractive indices. The depth of the focus can be adjusted manually relative to the cornea in a plane parallel to the corneal surface, to scan the cornea from epithelium to endothelium.
Confocal Imaging of Normal Cornea The HRT-RCM provides excellent resolution with sufficient contrast at different layers of the cornea, and distinct corneal layers from epithelium to endothelium can be visualized. The corneal epithelium is composed of five to six layers (Fig. 5(a)–(d)), as superficial cells, intermediate cells, and basal cells. The superficial cells are flat polygonal cells with well-defined border, prominent nuclei and uniform density of cytoplasm. It is about 40–50 mm in diameter and approximately 5 mm thick. The intermediate cells also known as wing cells are similar to superficial cells, but the nuclei are not evident. It is typically about 30–45 mm in size and characterized by the cell borders. Basal cells are approximately 10–15 mm and smaller in size. The basal cells appear denser than the superficial and wing cells. The subbasal nerve plexus (Fig. 5(e)) is characterized by the presence of hyper-reflective fibers of 4–8 mm length, and connected with anastomoses and organized in a vortex pattern in the lower nasal quadrant of the paracentral cornea. The visualization of corneal nerve plexus was not possible until the introduction of confocal microscopy. The nerve fibers appear bright and well contrasted against a dark background. Corneal stroma accounts for 90% of total corneal thickness and the keratocytes density is much higher in anterior stroma than in posterior stroma. The keratocyte counts are 1000 cells/mm2 in the anterior stroma and 700 cells/mm2 in the posterior stroma. The confocal image of the stroma exhibits multiple irregular oval, round or bean shaped bright structures representing keratocytes (Fig. 5(f) and (g)). The collagen fibers cannot be visualized by confocal microscopy in normal cornea. Corneal endothelium is composed of a single layer of cells that are 4–6 mm thick and approximately 20 mm in diameter. The endothelial cells progressively decline with age, and the normal endothelial cell count amounts to 3000 cells/mm2 in a healthy cornea. The endothelial cells appear as bright hexagonal and polygonal cells with unrecognizable nuclei (Fig. 5(h)).
Confocal Microscopy in Corneal Pathologies Confocal microscopy has been an effective tool in clinical diagnosis, and it is widely used to assess corneal infections, corneal dystrophies, pre and postsurgical assessment of laser-assisted in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), keratoconus and infectious keratitis. Also, it is used to monitor the cornea after injuries, and for differential diagnosis of
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Fig. 5 In vivo confocal microscopy images of the corneal layers in normal human subject from epithelium to endothelium. (a)–(d) Epithelium, (e) subbasal nerve plexus, (f) and (g) anterior and posterior stroma, and (h) endothelium. Courtesy of Prof. R. Guthoff, Prof. J. Stave, Dr. A. Zhivov, University of Rostock (from Heidelberg PowerPoint).
Fig. 6 In vivo confocal microscopy images demonstrating different forms of Langerhans cells. (a) Individual cell bodies without processes; (b) cells bearing dendrites; and (c) cells arranged in a meshwork via long interdigitating dendrites. Reproduced from Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Ophthalmology. Springer: Germany.
conjunctival tumors and conjunctivitis. As an example for clinical confocal imaging of the cornea, the Langerhans cell distribution and postsurgical assessment of LASIK using the HRT-RCM is shown in Figs. 6 and 7.
Langerhans Cells Langerhans cells can be evaluated using confocal microscopy with emphasis on cell morphology and cell distribution. In confocal microscopy, the Langerhans cells are visualized typically as bright corpuscular particles with dendritic morphology. These cells are distributed in low numbers at the center of the cornea and higher densities at the periphery of the cornea. Confocal microscopy helps in differentiating the Langerhans cells bodies that lack dendrites, Langerhans cells with small dendritic process form a local network, and Langerhans cells forming a meshwork via long interdigitating dendrites (Fig. 6(a)–(c)). The average density of Langerhans cells in normal subjects is 3473 cells/mm2 (range 0–64 cells/mm2) in the center of the cornea, and 9878 cells/mm2 (range 0–208 cells/mm2) in the periphery. Langerhans cells contribute to the immune and inflammatory responses, and determine the cell-mediated immunity.
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Fig. 7 Innervation after refractive surgery. (a) Nerve fibers near subbasal layer; (b) nerve fibers near Bowman’s membrane; (c) two months after LASIK – small regenerating nerve fibers in the central cornea; and (d) nine months after LASIK – new nerve fiber growing from the edge of flap region to center. Reproduced from Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Ophthalmology. Springer: Germany.
Postsurgical Assessment of LASIK LASIK is an excimer laser-based refractive surgical procedure that is currently being successfully used by surgeons to correct refractive errors (myopia, hyperopia etc.). After LASIK surgery, the cornea is typically evaluated by slit-lamp biomicroscopy and computerized corneal topography. The confocal microscopy evaluation of LASIK pre- and postsurgery adds new aspects in corneal diagnosis. The distribution of the corneal nerves and degeneration of the nerve structures can be visualized by confocal microscopy, and thus a direct comparison between innervation and sensitivity or symptom severity of the dry eye can be provided (Fig. 7).
Two-Photon Laser Scanning Microscopy Two-photon laser scanning microscopy based on the HRT was developed to investigate the cornea (Fig. 8). A compact femtosecond laser was employed in the system as the light source and an additional detection path for the two-photon mode was implemented. Both the confocal and two-photon images can be captured using the same instrument. An electronic switch facilitates the switching between the two imaging modes, while both detection modes are synchronized with the scanning unit. The laser beam is deflected in x- and y-direction via two scanning mirrors (scan unit, Fig. 8(b)) to trace out a square raster of 300 300 mm², 200 200 mm², or 150 150 mm² at a frame rate up to 16 Hz. The laser is focused on the cornea with a high numerical aperture objective lens (40 /0.80 NA or 16 /0.80 NA; interchangeable). The wide field of view can be achieved by using the 16 objective lens with less distinct cell morphology. Like confocal microscopy (HRT-RCM) the focus can be adjusted manually and different layers of the cornea can be visualized. A viscous sterile gel has to be placed between the objective lens and the cornea to eliminate the optical interface between two different indices.
Two-Photon Imaging of Normal Cornea The TPEF imaging of cornea has been extensively employed in preclinical studies, but has not been implemented in clinics yet. Although confocal microscopy has been used in clinical diagnosis, the collagen fiber orientation in the stroma cannot be visualized, and it provides only morphological information rather than the tissue’s functional state. With two-photon imaging
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Fig. 8 (a) Heidelberg Engineering two-photon microscopy and (b) schematic diagram of the optical set up of two-photon microscopy based on HRT. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Fig. 9 Two-photon imaging of corneal epithelial cells (a) and stromal collagen fibers (b) of human cornea. Reproduced from Qu, Y., Thomas, K. E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
both the tissue structures and the metabolic state can be well addressed. Two-photon microscopy provides excellent resolution and high contrast images of the cornea similar to confocal microscopy. High resolution two-photon images of the corneal epithelium and the collagen fibers from the human corneal buttons are shown in Fig. 9. The collagen fibers in the stroma can be well established with TPEF and SHG imaging.
Two-Photon Imaging of Limbal Stem Cells The limbal stem cells are located in the basal layer of the corneal limbus, and the proliferation of the limbal cells maintains the cornea by replacing the lost cells by tears. Limbal stem cells also prevent the conjunctival epithelial cells from migrating onto the surface of the cornea. The limbal stem cells have never been visualized with confocal microscopy and with the use of two-photon microscopy the limbal stem cells have been identified in mice. The limbal stem cells are smaller in size and it is shown by twophoton microscopy (Fig. 10). The two-photon imaging of limbal stem cells would help in the diagnosis of limbal stem cell deficiency and also differentiate and explore the nature of the limbal stem cells in a precise way.
Two-Photon Imaging of Infectious Keratitis Confocal microscopy plays a significant role in detecting the microorganisms causing corneal infections. It is difficult to identify the microorganisms like Acanthamoeba on the ocular surfaces since the appearance can impersonate as herpetic or bacterial keratitis. In most cases, the Acanthamoeba keratitis is diagnosed either by tissue culture or corneal biopsy, and histological analysis.
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Fig. 10 Two-photon imaging of mice limbal stem cells. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Fig. 11 Two-photon imaging of Acanthamoeba keratitis. Reproduced from Qu, Y., Thomas, K.E., Vola, M.E., et al., 2012. Rapid identification of microorganisms using the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 53 (14), 6175; Qu, Y., Thomas, K.E., Schuster, A.K., et al., 2011. Identification of microorganisms utilizing the two-photon ophthalmoscope. Investigative Ophthalmology and Visual Science 52 (14), 5823.
Although confocal microscopy lacks sufficient resolution in detecting the Acanthamoeba keratitis, it is used for diagnostic purposes since confocal microscopy is the only available diagnostic tool for patients. In confocal microscopy, the Acanthamoeba is visualized as round or ovoid highly reflective structures and the same is depicted by the two-photon microscopy (Fig. 11).
Second Harmonic Imaging of Cornea Collagen is the major component of connective tissues including cornea, and due to its transparency to visual and near infrared light the human eye is ideal for laser-based diagnostic and imaging applications. Among different multiphoton implementations, SHG imaging is particularly suitable to investigate non-centrosymmetric structures like collagen fibrils (Figs. 12 and 13). The basic structure of the collagen fibril is a triple helix composed of three proteins chains, which gives collagen the intrinsic ability of SHG
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Fig. 12 SHG imaging of corneal collagen fibrils in (a) forward and (b) backward directions from porcine cornea. The fibrillar structures resolved in (a) correspond to collagen bundles which are composed of regularly packed collagen fibrils. Scale¼10 mm. Reproduced from Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797.
Fig. 13 SHG imaging and two-photon imaging of human cornea from (a) Bowman’s membrane and (b) corneal stroma. The collagen fibers were visualized using the SHG imaging and the keratocytes (arrows) were visualized with the two-photon imaging. Reproduced from Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797.
imaging. Similar to TPEF, SHG is produced in only a small focal volume, permitting high resolution 3D optical sectioning of thick tissues. In contrast to TPEF, SHG from collagen is an intrinsic and a coherent process. Intrinsic imaging avoids the complications of slicing and labeling, and samples can be investigated under physiological conditions. Coherent constructive or destructive interference of SHG provides extra hints to the ultrastructure of collagen fibrils and their organizations. The collagen fibrils of the porcine cornea using SHG imaging are shown in Fig. 12. Most collagen fibrils run parallel to the cornea surface and share similar orientations with their neighbors. One of the unique features of SHG is that the geometry of the SHG emission field reflects the size and shape of the collagen fibrils. The SHG radiation pattern is mainly determined by the phase matching condition and the objects with the axial size on the order of the second harmonic wavelength exhibit forward directed SHG, while objects with a axial size less than l/10 (approx. 40 nm) are estimated to produce nearly equal backward and forward SHG signals. In Fig. 12(b), the backward SHG from cornea was extremely weak and there was no correlation between the structures revealed by forward and backward SHG imaging. The predominant forward SHG implies that the corneal collagen fibrils are not randomly distributed. It’s worth mentioning that the fibrillar structures revealed in Fig. 12(b) actually correspond to collagen bundles composed of many corneal collagen fibrils. In cornea, the collage fibrils (n ¼1.47) and the extrafibrillar matrix (n ¼ 1.35) have different refractive indices, thus the collagen fibrils have to be treated as individual scatters. Since the dimension of the collagen bundle (approximately 0.5 mm) is close to the second harmonic wavelength (400 nm), the phase correlation of SHG from neighboring collagen fibrils should be taken into account. The forward predominant SHG radiation from cornea indicates the regular arrangement of the collagen fibrils, at least in the domain of the collagen bundles. In Fig. 13, SHG and two-photon imaging of human cornea from Bowman’s membrane (Fig. 13(a)) and corneal stroma (Fig. 13 (b)) is depicted. The collagen fibers were visualized using the SHG imaging and the keratocytes (arrows) were imaged with TPEF imaging.
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Fig. 14 Two-photon microscopy of enucleated rabbit eyes 2 weeks after standard corneal CXL, upper row: two-photon imaging at 710 nm excitation light and lower row: second harmonic generation. (a) Two-photon imaging of the central zone of control cornea; (b) the peripheral zone of the treated cornea shows keratocytes in a dense network; (c) at the tangential zone, there is a sharp transition between the treated area and the untreated area, as shown by diminished keratocytes and a strong autofluorescence signal toward the central zone; (d) in the central zone, there are no keratocytes and the autofluorescence signal is uniform and strong; (e) and (f) second harmonic generation of stromal collagen in the control zone and the peripheral zone of the treated cornea shows a wavelike pattern; (g) at the transition zone toward the treated area, the wavelike pattern changes to a uniform signal; (h) in the central zone, a uniform SHG signal is visible. Reproduced from Steven, P., Hovakimyan, M., Guthoff, R.F., Hüttmann, G., Stachs, O., 2010. Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements. Journal of Cataract Refractive Surgery 36 (12), 2150–2159.
Two-Photon Imaging of Corneal Cross-linking Keratoconus is a progressive, noninflammatory and bilateral ectatic corneal disease characterized by corneal thinning and weakening. The thinning is most apparent at the apex of the cornea, and the steep conical protrusion leads to high myopia with irregular astigmatism. The visual loss occurs primarily from irregular astigmatism and myopia, and secondarily from the corneal scarring. Computerized corneal topography has become a gold standard for the early identification of keratoconus and recently confocal microscopy has also been used to assess the keratoconus cornea. Corneal cross-linking (CXL) is an invasive treatment procedure for keratoconus in order to stop or delay the process. The CXL procedure increases the mechanical and biomechanical stability of the cornea by application of ultraviolet-A light of 370 nm and riboflavin (vitamin B2). The assessment of cornea after cross-linking is limited because the treatment effect cannot be visualized by slit-lamp biomicroscopy. Confocal microscopy can detect the side effects like stromal edema and rarefication, the reappearance of stromal keratocytes over time. However, the effect of crosslinks between the collagen fibers after CXL cannot be visualized by confocal microscopy. Two-photon imaging with SHG is effective in measuring the crosslinks between collagen fibers as shown in Fig. 14.
Laser Safety Considerations of Two-Photon Microscopy The safe use of lasers has been a primary consideration since lasers are widely used for treatments and in research laboratories. The American National Standards Institute (ANSI) has specific standards for eye safety that take into account many of the important experimental variables such as laser power delivered at the cornea, viewing time, beam diameter or full-field viewing, the exposure area on the retina, and the wavelength. Near infrared radiation (NIR) relative to visible wavelength is considered to be less
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phototoxic and has deeper penetration in thick biological tissues, which is one of the key reasons for its widespread use in imaging devices. Compared to retinal imaging, the safety standards for illuminating the cornea and anterior structures of the eye are tolerant. The laser safety limits for corneal imaging can be calculated by the maximum permissible exposures (MPE) according to the ANSI standards as follows: Recommended MP corneal irradiance ¼ 25 t 0:75 W=cm2 for to10s Recommended MP corneal irradiance ¼ 4:0 W=cm2 for t410s For example, if the irradiated area is described by 1536 mm 1536 mm¼0.0236 cm2, the MPE should be 94.4 mW for longer exposure time (t410s) and should be 515 mW for shorter exposure time.
Future Directions Before implementing multiphoton microscopy for in vivo imaging of the human cornea, the shortcomings of these nonlinear microscopy techniques have to be addressed. For TPEF imaging, the drawback of depositing the energy into the tissue has to be evaluated to prevent photodamage. Although SHG and THG are absorption-free and can be used without photobleaching effects, harmonic imaging has another disadvantage that currently obstructs its use in ophthalmic practice. The harmonic signals are emitted predominantly in forward direction, whereas in backward direction the harmonic signals are weak. This limits SHG imaging for in vivo applications, and the backscattered signal might be enhanced using an absorbing dye. Also, new optical techniques such as wavefront aberration optimization and adaptive optics may be a key for future in vivo applications. Until these restrictions of multiphoton microscopy are eliminated, confocal microscopy remains exclusive in the assessment of cellular morphology in living human cornea.
Acknowledgment The authors like to thank Heidelberg Engineering GmbH (Heidelberg, Germany) for providing equipment support.
See also: Optical Coherence Tomography and Its Application to Imaging of Skin and Retina. Optics of the Human Eye
Further Reading Denk, W., Strickler, J.H., Webb, W.W., 1990. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76. Guthoff, R.F., Baudouin, C., Stave, J., 2006. Atlas of Confocal Laser Scanning In-Vivo Microscopy in Opthalmology. Germany: Springer. Guthoff, R.F., Zhivov, A., Stachs, O., 2009. In vivo confocal microscopy, an inner vision of the cornea – A major review. Clinical and Experimental Ophthalmology 37, 100–117. Han, M., Giese, G., Bille, J., 2005. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791–5797. Hao, M., Flynn, K., Nien-Shy, C., et al., 2010. In vivo non linear optical (NLO) imaging in live rabbit eyes using the heidelberg two-photon laser ophthalmoscope. Experimental Eye Research 91 (2), 308–314. Mantopoulos, D., Cruzat, A., Hamrah, P., 2010. In vivo imaging of corneal inflammation: New tools for clinical practice and research. Seminars in Ophthalmology 25 (5-6), 178–185. Steven, P., Hovakimyan, M., Guthoff, R.F., Hüttmann, G., Stachs, O., 2010. Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements. Journal of Cataract & Refractive Surgery 36 (12), 2150–2159.