RESUMEN
The morphology and the function of cellular and non-cellular structures in the living human cornea can be determined with modern correlative linear and nonlinear optical microscopic techniques and histology. Correlative microscopy is based on the use of different optical techniques to study the same specimen, ideally at the same location within the specimen, in order to increase the functional and/or morphological understanding of the specimen. A case study to assess the effect of overnight lid-closure on in vivo human corneal morphology is presented to illustrate correlative linear microscopy and optical low-coherence reflectometry. Nonlinear multiphoton excitation microscopy provides functional information on cellular metabolism based on the intrinsic fluorescence from the reduced pyridine nucleotides and the oxidized flavoproteins. Second-harmonic generation microscopy, a scattering process that does not deposit net energy into the tissue, provides structural information on corneal collagen organization. Molecular third-harmonic generation microscopy generates a signal in all materials and it an emerging technique. Coherent anti-Stokes Raman scattering microscopy provides chemical imaging for biology and medicine. The comparison and limitations of these microscopic modalities, linear and nonlinear microscopy applied to the cornea, and a review of some key findings is analyzed. A correlative integration and correlation of linear and nonlinear microscopies to study corneal function and structure is proposed to validate the clinical interpretation of microscopic images of the cornea.
Asunto(s)
Córnea/anatomía & histología , Microscopía/métodos , Lentes de Contacto/efectos adversos , Córnea/fisiología , Lesiones de la Cornea , Técnicas Histológicas , Humanos , Modelos Lineales , Microscopía/estadística & datos numéricos , Microscopía Confocal/métodos , Microscopía Confocal/estadística & datos numéricos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/estadística & datos numéricos , Dinámicas no Lineales , Espectrometría Raman/métodos , Tomografía de Coherencia Óptica/métodos , Tomografía de Coherencia Óptica/estadística & datos numéricosRESUMEN
Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.
Asunto(s)
Quemaduras/etiología , Quemaduras/prevención & control , Microscopía de Fluorescencia por Excitación Multifotónica/efectos adversos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Piel/patología , Piel/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Rayos Infrarrojos/efectos adversos , Rayos Láser/efectos adversos , Dosis de Radiación , Medición de Riesgo/métodos , Factores de Riesgo , Piel/lesiones , Técnicas de Cultivo de TejidosRESUMEN
The retinal circulation of the normal human retinal vasculature is statistically self-similar and fractal. Studies from several groups present strong evidence that the fractal dimension of the blood vessels in the normal human retina is approximately 1.7. This is the same fractal dimension that is found for a diffusion-limited growth process, and it may have implications for the embryological development of the retinal vascular system. The methods of determining the fractal dimension for branching trees are reviewed together with proposed models for the optimal formation (Murray Principle) of the branching vascular tree in the human retina and the branching pattern of the human bronchial tree. The limitations of fractal analysis of branching biological structures are evaluated. Understanding the design principles of branching vascular systems and the human bronchial tree may find applications in tissue and organ engineering, i.e., bioartificial organs for both liver and kidney.
Asunto(s)
Fractales , Retina/fisiología , Animales , Fenómenos Biomecánicos , Bronquios/patología , Difusión , Humanos , Pulmón/patología , Modelos Anatómicos , Retina/embriología , Retina/metabolismo , Ingeniería de TejidosRESUMEN
This paper explores the seminal contributions of David Maurice to the field of ophthalmic instrumentation. His development of the specular microscope, the scanning slit optical confocal microscope, and the corneal microfluorometer resulted in advances in our understanding of corneal morphology, physiology, and pathology. The development of the scanning slit, clinical confocal microscope is not a new paradigm or a paradigm shift, but a continuous series of interlinked technical advances from the early work of Vogt to Thaer's development of a clinical confocal microscope. For each instrument both the connection to the prior work of others and the unique advances are discussed and contrasted. This paper develops the connections and parallel developments in the instrument developments of Goldmann, Maurice, Svishchev, Baer, Koester, Masters, and Thaer. The evidence in support of the thesis consists of published papers, patents, personal communication, and study of Goldmann's book collection in Bern. A second theme is that knowledge of physics is a prerequisite for optical instrument development in ophthalmology. David Maurice had a university degree in physics and Hans Goldmann learned physics from his books. The contributions of David Maurice to optical instrumentation follow the major contributions of Goldmann and facilitated and stimulated other scientists who acknowledged their important intellectual debt to David Maurice.
Asunto(s)
Técnicas de Diagnóstico Oftalmológico/historia , Técnicas de Diagnóstico Oftalmológico/instrumentación , Microscopía Confocal/historia , Córnea/anatomía & histología , Diseño de Equipo , Historia del Siglo XX , Humanos , Londres , Microscopía Confocal/instrumentaciónRESUMEN
In 1931, Maria Göppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria Göppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.
Asunto(s)
Microscopía Confocal/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Fotones , Historia del Siglo XX , Microscopía Confocal/historia , Microscopía de Fluorescencia por Excitación Multifotónica/historia , Sensibilidad y EspecificidadRESUMEN
Three-dimensional confocal microscopy of the living eye is a major development in instrumentation for biomicroscopy of the eye. This noninvasive optical technology has its roots in the application of optics to reflected light imaging of the eye. These instrument developments began with Leeuwenhoek's use of his single lens microscope to investigate the structure of the eye. There followed a series of connected instruments: the ophthalmoscope, the slit lamp, the specular microscope, and the clinical confocal microscope. In vivo confocal microscopy produces high contrast, reflected light images or optical sections through the depth of living ocular tissue. Stacks of registered optical sections can be transformed by computer visualization techniques into three-dimensional volume images of ocular tissues: cornea, ocular lens, retina, and optic nerve. The clinical confocal microscope has resulted in new diagnostic techniques and new cellular descriptions of ocular disorders and pathology.