La microscopía confocal de reflectancia in vivo (MCR) es una técnica no invasiva que permite obtener un diagnóstico de forma inmediata y en tiempo real de patología cutánea tumoral con una precisión diagnóstica que se acerca al diagnóstico histológico convencional. La MCR puede permitir un seguimiento clínico de respuesta a tratamientos no invasivos de patología tumoral, como la terapia fotodinámica, sin necesidad de realizar biopsias. El presente trabajo reúne cuatro estudios realizados con el fin de demostrar la utilidad de la técnica de MCR en el estudio y manejo clínico de las neoplasias cutáneas. En el primer estudio se incluyeron de forma prospectiva 154 tumores cutáneos para establecer qué parámetros de microscopía confocal se relacionaban con cada tumor, analizar si estos criterios eran reproducibles y finalmente evaluar la correlación de estos parámetros con la dermatoscopia y la histología convencional. Según los resultados de este primer trabajo, cuatro características observables mediante MCR permitían diferenciar las lesiones melanocíticas (LM) de las no melanocíticas (LNM): patrón epidérmico en empedrado, crecimiento pagetoide, presencia de nidos celulares en la dermis y presencia de papilas dérmicas bien definidas en toda la lesión. Dentro de las LM, la presencia de células redondas en estratos suprabasales y de células nucleadas atípicas en la dermis se asociaban al diagnóstico de melanoma; mientras la presencia de papilas dérmicas con contorno reflectante (anillos basales) así como la observación de células basales típicas, se asociaban a nevus. Basándonos en la reproducibilidad y correlación histológica y dermatoscópica de los criterios de MCR, se desarrolló un algoritmo en dos etapas para el diagnóstico de melanoma consiguiendo una sensibilidad del 86,1% y una especificidad del 95.3% (J Am Acad Dermatol. 2009;61:216-29). En el segundo trabajo, se estudiaron los parámetros de microscopía confocal en melanomas de extensión superficial (MES) en fase de crecimiento vertical y en melanomas nodulares (MNs). Para ello se incluyeron de forma prospectiva 10 MNs, 10 MES con área nodular y 10 MES con área palpable. Se realizó un análisis sistemático y estudio estadístico de las características dermatoscópicas, confocales e histológicas de los 3 grupos de lesiones. Mientras los MNs mostraban patrones de dermatoscopia inespecíficos, los MES exhibían patrones multicomponente y puntuaciones más altas en los algoritmos de dermatoscopia. Por MCR, los MNs tenían poco crecimiento pagetoide y presentan a menudo un patrón normal de la epidermis, a diferencia de los MES que se caracterizaban por un patrón desestructurado de la epidermis y la presencia de abundantes células en estratos suprabasales Tanto en los MNs como en las áreas nodulares de los MES, en la unión dermo-epidérmica no se visualizaban las papilas dérmicas, en su lugar se observa una proliferación de células reflectantes atípicas no agregadas. En la dermis, los MNs exhibían a menudo unos agregados celulares característicos de aspecto cerebriforme, que podían observarse también en las áreas nodulares de algunos MES (Arch Dermatol. 2008;144:1311-20). En un tercer artículo estudiamos las características del carcinoma basocelular pigmentado (CBC) mediante MCR, histología e inmunohistoquímica (S-100, MelanA, HMB-45 y CD1a).Mediante MCR demostramos la presencia de estructuras reflectantes muy características de aspecto dendrítico dentro de los nidos tumorales situados en la dermis de los CBC estudiados, que correspondían por estudio inmunohistoquímico a melanocitos no neoplásicos que poblaban los nidos (Arch Dermatol. 2007;143:883-6). Finalmente, en un cuarto trabajo estudiamos la aplicabilidad de la MCR en el diagnóstico y seguimiento de pacientes con genodermatosis de alto riesgo de desarrollar cáncer cutáneo (síndrome de Gorlin -SG- y Xeroderma pigmentoso -XP-) afectos de CBCs múltiples y tratados con terapia fotodinámica con metil-aminolevulinato (MAL). Se incluyeron 4 pacientes con SG y 2 hermanos con XP. Se trataron lesiones únicas o múltiples en zonas localizadas con 1 a 3 ciclos de TFD-MAL. La exploración con MCR se realizó antes y 3 meses después del tratamiento en las lesiones diana. Se trataron 13 CBCs pigmentados faciales en los pacientes afectos de XP y múltiples lesiones en cara o tronco (hasta 200) en los afectos de SG. Globalmente se obtuvo una remisión clínica completa en un 25- 67% de las lesiones. La respuesta al tratamiento puedo ser correctamente evaluada mediante MCR (J Eur Acad Dermatol Venereol. 2011;25:819-27). En conclusión, la MCR parece ser útil en el diagnóstico diferencial de las lesiones pigmentadas y complementa a la dermatoscopia en el diagnóstico del melanoma. Esta técnica permite caracterizar subtipos de melanoma como el melanoma nodular. También sirve de ayuda en el diagnóstico y caracterización del carcinoma basocelular pigmentado y en su monitorización tras tratamiento con terapias no invasivas como la terapia fotodinámica. / This work brings together four studies to demonstrate the clinical utility of reflectance confocal microscopy (RCM) in the clinical management of cutaneous neoplasms. In the first study we prospectively included 154 skin tumors in order to develop an algorithm for the in vivo diagnosis of skin tumors. We analyzed RCM features on stored images before excision and performed statistical analyses to determine the association of RCM features with tumor types. Four confocal features differentiated melanocytic lesions (ML) from non-melanocytic lesions (NML): cobblestone pattern of epidermal layers, pagetoid spread, mesh appearance of the dermoepidermal junction, and the presence of dermal nests. Within ML, the presence of roundish suprabasal cells and atypical nucleated cells in the dermis was associated with melanoma, and the presence of edged papillae and typical basal cells was associated with nevi. Based on the reproducibility and histological and dermoscopic correlation of MCR criteria, we developed a two-step algorithm for the diagnosis of melanoma, achieving a sensitivity of 86.1% and specificity of 95.3% (J Am Acad Dermatol. 2009; 61: 216-29). In the second work we characterized nodular melanoma (NM) by dermoscopy, RCM and histopathology. We included 10NMs, 10 superficial spreading melanomas (SSMs) with a nodular area, and 10 SSMs with a blue palpable area but not yet nodular. Whereas NMs had predominantly nonspecific global dermoscopic patterns, SSMs exhibited a multicomponent pattern and higher dermoscopic scores. By RCM, distinctive confocal features were observed at all cutaneous levels in NMs compared with SSMs, suggesting different biological behavior. (Arch Dermatol. 2008, 144:1311-20). In the third article we explored the confocal features of pigmented basal cell carcinoma (BCC) and correlated the findings with histology and immunohistochemistry. RCM revealed highly refractive dendritic structures within tumor nests that corresponded to the presence of melanocytes within the tumor. RCM allowed the study of pigmented BCC and the identification of specific criteria. (Arch Dermatol. 2007; 143:883-6). Finally, in the fourth paper we aimed to evaluate the efficacy of methyl-aminolevulinate (MAL) photodynamic therapy (PDT) in basal cell carcinomas (BCCs) from patients with Gorlin syndrome (GS) and Xeroderma pigmentosum (XP), and to determine the utility of RCM in the diagnosis and the evaluation of therapeutic response. We included 4 patients with GS and 2 siblings with XP. Single or multiple lesions in localized areas were treated with 1 to 3 cycles of MAL PDT. RCM was performed before and 3 months after the treatment in target lesions. Overall, we obtained a complete clinical remission in 25 to 67% of the lesions. RCM could identify confocal features for BCC and assess tumor remissions after treatment (J Eur Acad Dermatol Venereol. 2011; 25:819-27).

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Although the effects of diabetes on bone mineral content has been studied, little is known about the structural alterations in collagen, maturation of apatite crystals and carbonate content in diabetic bone. The first part of this study aimed to investigate the mineral and organic properties of cortical, trabecular and growth plate regions of rat femur tissues in type I diabetes using FTIR microspectroscopy and Vickers microhardness test. A decrease in mineral content (degree of mineralization), decrease in microhardness, increase in carbonate content, increase in size and maturation of hydroxyapetite crystals, which are the implications of increased osteoporosis, were observed in diabetic bone. In addition, a decreased carbonate substitution into bone apatite and an increase in labile type carbonate was observed in diabetic bone. There was a decrease in the level of crosslinking of collagen in cortical and trabecular regions of diabetic femurs, implying a decrease in bone collagen quality that may contribute to bone fragility. Recent evidence implies that intramyocellular lipid accumulation is directly correlated with insulin resistance, a key parameter in the generation of obesity. The second part of this study is mainly focused on the determination of the structural and compositional characterization of macromolecules of longissimus dorsi and quadriceps muscles of Berlin fat mouse inbred (BFMI) lines using ATR-FTIR spectroscopy and FTIR microspectroscopic imaging, together with the quantification of fiber specific distribution of lipids in these muscles by the use of confocal microscopy. The study groups included 10 weeks old standard breeding diet fed (juvenile) and 20 weeks old high fat diet fed control and BFMI lines. The results revealed the loss of unsaturation in lipids, increased triglyceride content, increased amount of lipids having shorter chain length, increased lipid peroxidation and fiber specific accumulation of lipids in type IIa and intermediate fibers in skeletal muscles of both 10 weeks old and 20 weeks old BFMI lines, emphasizing their obese phenotype. However, the alterations were more prominent in skeletal muscles of 20 weeks old high fat diet fed BFMI lines, displaying a more severe obesity phenotype. The results of the characterization revealed that BFMI860 and BFMI861 lines are convenient models for the study of spontaneous obesity and studies to enlighten the genetic basis of obesity.

The capability to resolve the contributions from spectroscopically overlapping fluorophores has enabled significant breakthroughs in cellular imaging. However, commercial microscopes for this purpose use analog light detection with least squares curve-fitting analysis and improvements in sensitivity are needed. To this end, a microscope has been constructed with high throughput and single-photon detection capability. The fluorescence is separated through use of a prism spectrometer or a series of dichroic mirrors into four spectral bands and detected using four single-photon avalanche diode (SPAD) detectors, which provide high-quantum efficiency in the red spectral region. The detectors are connected to a time-correlated single photon counting module to provide sub-nanosecond temporal resolution for distinguishing fluorophores with different fluorescence lifetimes. Maximum-likelihood (ML) methods have been developed for analyzing the temporally and spectrally resolved photon count data from the SPADs to find the contributions from different fluorescent species and from background. Commercially available SPADs exhibit a count-rate dependent time shift in the impulse response function, and hence the instrument incorporates custom modified SPADs with improved timing stability. Nevertheless, there is still some time shift, and hence the ML-analysis has been extended to include this as an adjustable parameter for each individual SPAD. Monte Carlo simulations have also been developed to enable studies of the number of photons needed to resolve specific fluorophores.

Cancer is a world menace. After years of endeavor seeking the end of it, people started to realize that no matter how powerful the therapy could be, detection at early stage is always a cheaper, easier and more successful solution compared with curative methods for cancer developed onto its advanced stage. However, relatively few early-detection approaches have proven sufficiently effective and practical for mass use as a point-of-care tool. An early-cancer screening tool integrating the desired features of sensitive, informative, portable, and cost-effective is in need for the doctors. The progress in optical imaging and Micro-electro-mechanical system (MEMS) technology offers a promise for an innovative cancer screening alternative that is non-invasive, radiation-free, portable and potentially cost-effective. This dissertation investigates handheld instrumentation as multi-modalities of miniature imaging probes with various designs of MEMS devices, to obtain real-time images of epithelial tissue optical and physiological properties, combining the quantitative advantages of spectral analysis with the qualitative benefits of imaging to distinguish early cancer. This dissertation in sequence presents the handheld instruments in the fashions of Laser-scanning confocal microscopy (LSCM), optical diffuse reflectance imaging, nonlinear optical imaging modalities with their subsequent image-guided managements in oral cancer, skin cancer detection, circulating tumor cell (CTC) imaging, and imaging guided surgeries. One of the main challenges facing miniaturization lies in the mechanism of beam deflection across the sample. This dissertation introduces two generations of MEMS devices desgined, fabricated and incorporated in the imaging probes. A two-axis vertical comb driven silicon micromirror was used in the development of a handheld LSCM for oral cancer detection. Though obtaining numerous advantages, this first generation silicon MEMS micromirror suffers from small aperture size and high voltage requirement for actuation, which result in low collection efficiency in fluorescence imaging and medial safety concerns, respectively. Therefore a stainless steel scanner compatible with electrical discharge machining (EDM) process was fabricated with simplified process, low-voltage magnetic actuation and large fluorescence collection efficiency, with its capability demonstrated in the incorporation and embodiment of a handheld hyperspectral nonlinear imaging probe. Besides, software and controlling innovations for handheld imaging modalities are presented. A feedback controlling system for MEMS scanning status monitoring was developed for stabilized imaging rendering. For the sake of further improved imaging stability in handheld imaging and to enable on-site mosaic for large field viewing, a handheld mosaic system was developed and presented. / text

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

[Truncated abstract] An important field of research in orthopaedic biomechanics is the elucidation and mathematical modelling of the mechanical response of cartilaginous tissues. Such research has applications in the understanding of joint function and degenerative processes, as well as in surgical planning and simulation, and engineering of tissue replacements. In the case of surgical and tissue engineering applications especially, patient-specific mechanical properties are highly desirable. Unfortunately, obtaining such information would generally involve destructive mechanical testing of patient tissue, thus rendering the tissue functionally unusable. Development of a laser scanning confocal arthroscope (LSCA) within our School will soon allow non-invasive extraction of 3D microstructural images of cartilaginous tissues in vivo. It is also envisaged that, linked to a suitably formulated constitutive formulation, such information could allow estimation of tissue mechanical response without physical biopsy. This thesis describes the development of techniques to potentially allow non-invasive patient-specific estimation of tissue mechanical response based on confocal arthroscopy data. A microstructural constitutive model is developed which is capable of directly incorporating LSCA-derived patient-specific structural information. A fibre composite type homogenisation approach is used as the basis for the model. ... The result is a series of orientation tensors describing the 3D orientation of linear features in the image stack. The developed analysis techniques are used to estimate fibre volume fraction and orientation distribution for each of the meniscal specimens. The developed constitutive model and image-derived structural parameters are finally used to estimate the reaction force history of two meniscal cartilage specimens subjected to partially confined compression. The predictions are made on the basis of the specimens? individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework. The likely limitations and potential areas of improvement are discussed.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.