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This paper reviews the literature from 1990 to 2000 and evaluates the seminal investigations performed with the confocal microscope on the in vivo human cornea. Our pedagogical technique is to illustrate both the advantages and the problems associated with occular confocal microscopy by way of annotated examples. Confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level. The ability to provide high-resolution, real-time images of the full thickness of the cornea gives the clinician and the researcher an important new tool for the investigation of the cornea.

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In vivo, scanning-slit, confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level. The ability to provide high resolution, real-time images of the full thickness of the living human cornea gives the clinician and the researcher an important new tool.

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Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.

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This paper provides the clinician and the researcher with an in-depth manual on the use of a scanning-slit confocal light microscope for the clinical examination and investigation of the living human cornea in vivo. The scope of the paper includes a thorough explanation of the principles of various types of confocal microscopes as well as their limitations, a comprehensive review of the development of biomicroscopy of the eye, and a comparison of confocal microscopy and other optical techniques such as optical coherence tomography. The early work of Ridley, Goldmann and others on point illumination in early confocal instruments is described. The main part of the paper describes and illustrates the clinical examination of the living human cornea with the confocal microscope. Figures (many in color) from the normal cornea, the cornea with known parthologies, and the postsurgical cornea are selected for their educational value. Photographs of standard light microscopy of fixed, human corneal sections are compared with confocal maicroscopic images. Where appropriate, slit lamp color photographs are compared with confocal microscopic images. The clinical scanning-slit confocal microscope is an evolving instrument for biomicroscopy of the living eye.

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This study investigates the precision and intraindividual variability of a clinical optical pachometer based on low-coherence reflectometry, which was used to measure the central thickness of a human cornea in vivo. The instrument, attached to a slit lamp, is a single mode fiber optic based Michelson interferometer with a high repetition rate as previously described. The same operator performed ten sets of measurements on the same subject, each consisting of 20 consecutive scans, on each day for three consecutive days. By computing the means from every scan series, the thickness of the central cornea with optical pachometry was found to be 519.6±1.2 (range 518-521) µm on day 1, 519.9±0.9 (range 519-521) µm on day 2, and 523.8±0.6 (range 523-525) µm on day 3. The thickness values on day 3, where the subject suffered from a cold without clinical ocular involvement, were different from the two previous days (p<0.001, one way analysis of variance). Optical low-coherence reflectometry measurements of corneal thickness can be performed with high precision of about 1 µm and a high intra- and intersession reproducibility. © 1999 Society of Photo-Optical Instrumentation Engineers.

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This paper explains the term low-coherence interferometry, reviews the early development of optical low-coherence reflectometry, and shows some of the paths that led to the field of biomedical optics. This paper demonstrates that early technical developments in the telecommunications industry resulted in a myriad of technical implementations and applications in biology, medicine, and the explosion of the field in noninvasive biomedical optical techniques. Recent examples of innovative applications of this proliferating technology into the fields of ophthalmology, developmental biology, and endoscopy are described. © 1999 Society of Photo-Optical Instrumentation Engineers.

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Two-photon excitation microscopy has the potential as an effective, noninvasive, diagnostic tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue scattering and absorption coefficients in the infrared wavelengths. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multiphoton excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 200 microns. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical biopsy.

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OBJECTIVE: Confocal in vivo real-time microscopy was applied to study the corneal morphology in long-term contact lens wearers. DESIGN: In a cross-sectional study, patients with a history of long-term contact lens wear underwent corneal confocal microscopy. The authors investigated 13 patients with a history of up to 26 years of soft contact lens wear, 11 patients with a history of up to 25 years of rigid gas permeable contact lens wear, and a control group of 29 normal subjects without a history of contact lens wear. INTERVENTION: Scanning slit-confocal microscopy was performed with a 50x/1.0 NA water immersion objective. Corneal optical sections were recorded in real time without further digital processing and reviewed frame by frame. MAIN OUTCOME MEASURES: Video frames selected from all corneal layers were evaluated qualitatively. The new finding of panstromal microdot deposits was quantitated in a scoring system ranging from 0 to 4+. Corneal endothelial cell densities were counted with the fixed frame technique. RESULTS: Epithelial microcystic changes and alterations of endothelial cell morphology were found to a variable extent as described previously. A new finding was there were highly reflective panstromal microdot deposits in the corneal stroma. For this new disease, a scoring system ranging from 0 to 4+ was established. In the control group, 0 of 29 patients had stromal microdot deposits. In the soft contact lens group, 13 of 13 patients had panstromal microdot deposits with a mean score of 3.1 (range, 1-4), and in the hard contact lens group, 11 of 11 had a mean score of 1.9 (range, 1-4) for corneal microdot deposits. CONCLUSIONS: With confocal microscopy, a new type of chronic stromal change has been observed in all subjects with long-term contact lens wear. Because subjects with soft contact lens wear had a more pronounced corneal degeneration than did subjects with gas permeable lenses, the authors assume the deposits to be induced by chronic hypoxia. The condition of stromal microdot degeneration as observed with confocal microscopy may be the early stage of a significant corneal disease, which eventually may affect large numbers of patients after decades of contact lens wear.

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Fourier analysis of in vivo human corneal endothelial cell structure was investigated using specular photomicrographs for a range of ages from less than one year to over 70. The theoretical basis for this analysis was investigated using mathematical models of cell structures where the elements determining their form could be modified in a controlled and quantified manner. The resulting Fourier transform properties were related to properties of cell structure. The experimental factors underlying this analysis were then studied using digitized images of corneal endothelial cells. It was found that the Fourier transforms provided quantitative descriptions of population cell size and organisation. For the smaller, more regular cell structure from the younger eyes, the expected larger rings of the Fourier transforms were demonstrated. Specular photomicrographs of older eyes gave rise to smaller diameter rings in their Fourier transforms. These results are consistent with the previous studies which used manual tracings of human endothelial cell patterns. This is the first demonstration of the direct Fourier analysis of clinical human corneal specular photomicrographs.

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Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.

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A technique that transforms a set of images acquired as a rotating frame about an axis (Z axis) into a set of images along the Z axis in presented. This technique is applied to the three-dimensional visualization of the in vivo human lens. A Scheimpfling slit camera acquired 60 optical images through the in vivo human lens. Between each image acquisition the plane containing the slit beam of light was sequentially rotated. This set of 60 images was transformed into a new stack of images on the Z axis. The transformed stack of Z images was visualized with volume rendering software.

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The potential of confocal light microscopy (CLM) for in vivo observation of pathology in the anterior pole of the eye lenses was evaluated by performing an in vitro study of human lenses comparing this type of microscopy with scanning electron microscopy (SEM). In vitro CLM showed high resolution images of the epithelium which would enable early detection of pathology and easily allows cell counting and estimating cell size. Superficial lens fibres are well visualised and low and high frequency bands as well as vacuolar elements were easily detected. SEM observations fully supported the CLM observations. This study shows that CLM has the potential to become a useful tool for detecting lens changes, after suitable adaptation for clinical use.

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A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 microns with a vertical resolution of 2 microns. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-micron vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.

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A series of 60 reflected-light, rotating slit images of the human cornea in vivo are acquired with a rotating Scheimpflug camera. Each single acquired optical slice on a single meridian contains the shape of the anterior and posterior surfaces of the cornea. These 60 images are mathematically transformed into a three-dimensional volume that can be visualized on any meridian, including those images on meridians that have not been selected during the image-acquisition process. The optical distortions and aberrations of both the camera system and the eye are not included in this study using a rotating slit camera to determine the shape of both anterior and posterior corneal surfaces. The method presents the view of the anterior and posterior cornea (out to the midperipheral region) and a reference plane of the iris reflection. Further development of this technique may find clinical applications in corneal refractive surgery.

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A human lens containing a cataract has been visualized in vivo by a computer transformation of a rotated set of Scheimpflug digital-image slices through the three-dimensional volume of the lens. At each angular position (incremented 3 degrees about the optical axis) of the camera a digital image of the ocular lens in vivo was acquired. Data acquisition was made with a series of 60 Scheimpflug images. The set of optical sections were aligned to correct for small eye movements during the data-collection process prior to computer transformation into a new set of slices, which are orthogonal to the optical axis of the eye. The use of slices orthogonal to the optical axis to visualize lenticular light scatter represents a new, simple technique, which can be performed on a personal computer to visualize in vivo human cataracts in three dimensions.

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