Development of a new spectral imaging system for the diagnosis of skin cancer

The incidence of skin cancer in Europe, US and Australia has been rising rapidly. Skin cancer accounts for one in three cancers worldwide and a person has 1:25 chance to develop a melanoma, the most aggressive form. Visual inspection followed by histological examination is, still today, the gold standard for clinicians, which is carried out through a dermoscope, a handheld device with a magnifying lens and a white and uniform illumination field. The dermoscopic technique requires considerable training in the interpretation of what is seen and is highly dependent on subjective impressions. In consequence, a large number of unnecessary surgical procedures are performed. For this reason, in this thesis a spectral imaging system to improve skin cancer diagnosis has been developed. This work has been carried out in the framework of the European project DIAGNOPTICS "Diagnosis of skin cancer using optics", which aimed to launch a hospital service based on a multiphotonic platform to improve skin cancer with the combination of four non-invasive novel techniques: 3D and multispectral imaging, optical feedback interferometry and confocal microscopy. The handheld system built included a monochromatic CCD camera attached to an objective lens and a light source containing 32 light emitting diodes (LEDs) with 8 spectral bands from 400 nm to 1000 nm. An acquisition software to control all the components of the multispectral system was programmed as well as a simplest version for physicians. The changes over time of the emission of the LEDs was analysed, and also the linear response of the camera at each wavelength, the uniformity of the LED emission and the short and long-term repeatability of the system in acquiring images, to ensure the good performance of the system. In order to proceed with the Ethical Committee approval and to launch the systems in both hospitals, irradiance and radiance measurements were done according to the standard UNE-EN 62471. A Graphical User Interface (GUI) was developed for the spectral images processing and corresponding analysis, allowing spectral and colorimetric features to be computed in terms of reflectance, absorbance and colour parameters. Furthermore, a segmentation algorithm was also implemented to extract the isolated information from the lesion. For all images calculated in terms of any of the parameters, conventional statistical descriptors were obtained. As a first approach to extracting textural information we also used the analysis of the statistical properties of the histogram. An inclusion criteria and a measurement protocol were established. From all lesions analysed, 620 were measured with the multispectral system, 572 of them had a clinical or histopathological diagnosis, and 502 could be properly segmented. Therefore, 429 skin lesions were finally included in the study: 290 nevi, 95 melanomas and 44 basal cell carcinomas. A classification algorithm was developed in order to decide whether the lesions were malignant (melanomas and basal cell carcinomas) or not (nevi), splitting previously the data into training and validations set of the same size. 15 parameters from 1309 were found to be not redundant providing a 91.3% of sensitivity and 54.5% of specificity. Accordingly, the addition of textural information was shown to be useful for the diagnosis of malignant lesions than the sole use of averaged spectral and colour information. The same steps were carried out for the 3D imaging system also included in the multiphotonic platform. In this case, 3 parameters were found to be useful for the classification providing values of 55.6% and 83.7% of sensitivity and specificity, respectively. Finally, the combination of both system was also tested as a first attempt to improve the detection of melanomas, providing 100% and 72.2% of sensitivity and specificity, respectively. However, the conclusions reached in this case should be taken with caution due to the limited number of lesions.La incidència del càncer de pell a Europa, Estats Units i Austràlia ha anat augmentant ràpidament. Representa un de cada tres càncers a tot el món i una persona té 1:25 oportunitats de desenvolupar un melanoma, la forma més agressiva. Actualment, la inspecció visual amb un dermoscopi seguida d'un examen histològic és l'estàndard utilitzat pels metges a l'hora de diagnosticar-lo. La dermoscòpia requereix una formació considerable per interpretar el que es veu i depèn de les impressions subjectives dels clínics. En conseqüència, es realitzen una gran quantitat de procediments quirúrgics innecessaris. Per aquest motiu, en aquesta tesi s'ha desenvolupat una sistema d'imatge espectral per millorar el diagnòstic del càncer de pell. Aquest treball s'ha realitzat dins el marc del projecte Europeu DIAGNOPTICS ¿Diagnosis del càncer de pell utilitzant òptica?, el qual ha posat a punt un servei hospitalari basat en un plataforma multifotònica que combina quatre tècniques òptiques innovadores: sistemes d'imatge multiespectral 3D, interferometria de retroalimentació i microscòpia confocal. El sistema portàtil desenvolupat inclou una càmera monocromàtica CCD, un objectiu i una font de llum formada per 32 díodes emissors de llum (LED) amb 8 bandes espectrals diferents que emeten des de 400 nm fins a 1000 nm. S'ha preparat un programa d'adquisició per controlar tots els components del sistema així com una versió més simple del mateix pels metges. Per tal d'assegurar el bon funcionament del sistema, es van analitzar els canvis temporals en l'emissió dels LEDs així com la seva uniformitat d'emissió, la resposta lineal de la càmera per cada longitud d'ona i la repetibilitat del sistema pel què fa a l'adquisició d'imatges. Per tal d'obtenir l'aprovació del Comitè Ètic i poder realitzar l'estudi clínic en els hospitals, es van dur a terme mesures d'irradiància i radiància d'acord amb la norma UNE-EN 62471. També es va implementar una interfície gràfica d'usuari (GUI) per al processament de les imatges espectrals i la seva corresponent anàlisi. Aquest algorisme permet calcular paràmetres espectrals i colorimètrics de la pell en termes de reflectància, absorbància i d'altres basats en el color. A més, inclús es va desenvolupar un algorisme de segmentació per extreure informació aïllada de cada lesió. Per a totes les imatges calculades en termes de qualsevol paràmetre, es van obtenir descriptors estadístics convencionals i també es van utilitzar propietats estadístiques dels histogrames com una primera aproximació d'extreure informació de textura de la lesió. Finalment, es van establir els criteris d'inclusió i un protocol de mesura. De totes les lesions analitzades, se'n van mesurar 620, de les quals 572 tenien un diagnòstic clínic o histopatològic; 502 es van poder segmentar adequadament. D'aquesta manera es van incloure 429 lesions cutànies a l'estudi: 290 nevus, 95 melanomes i 44 carcinomes de cèl·lules basals. Es va desenvolupar un algorisme de classificació per determinar si les lesions eren malignes (melanomes i carcinomes de cèl·lules basals) o no (nevus), dividint prèviament les dades en un grup d'entrenament i un altre de validació de la mateixa mida. Es va observar que 15 del 1309 paràmetres proporcionaven informació rellevant per a la classificació, obtenint una sensibilitat del 91,3% i una especificitat del 54,5%. Així doncs, es demostra que la incorporació d'informació de textura és molt útil per al diagnòstic del càncer de pell més enllà de la informació espectral i de color. Aquests mateixos passos es van seguir pel sistema 3D també inclòs en la plataforma multifotònica, tot i que en aquest cas el número de lesions de què es disposava era més limitat. En aquest cas, es van seleccionar 3 paràmetres i es va obtenir una sensibilitat del 55,6% i una especificitat del 83,7%. Finalment, amb la combinació d'ambdós sistemes la sensibilitat obtinguda va ser de100% i l'especificitat del 72,2%.Postprint (published version

Development of a new spectral imaging system for the diagnosis of skin cancer

The incidence of skin cancer in Europe, US and Australia has been rising rapidly. Skin cancer accounts for one in three cancers worldwide and a person has 1:25 chance to develop a melanoma, the most aggressive form. Visual inspection followed by histological examination is, still today, the gold standard for clinicians, which is carried out through a dermoscope, a handheld device with a magnifying lens and a white and uniform illumination field. The dermoscopic technique requires considerable training in the interpretation of what is seen and is highly dependent on subjective impressions. In consequence, a large number of unnecessary surgical procedures are performed. For this reason, in this thesis a spectral imaging system to improve skin cancer diagnosis has been developed. This work has been carried out in the framework of the European project DIAGNOPTICS "Diagnosis of skin cancer using optics", which aimed to launch a hospital service based on a multiphotonic platform to improve skin cancer with the combination of four non-invasive novel techniques: 3D and multispectral imaging, optical feedback interferometry and confocal microscopy. The handheld system built included a monochromatic CCD camera attached to an objective lens and a light source containing 32 light emitting diodes (LEDs) with 8 spectral bands from 400 nm to 1000 nm. An acquisition software to control all the components of the multispectral system was programmed as well as a simplest version for physicians. The changes over time of the emission of the LEDs was analysed, and also the linear response of the camera at each wavelength, the uniformity of the LED emission and the short and long-term repeatability of the system in acquiring images, to ensure the good performance of the system. In order to proceed with the Ethical Committee approval and to launch the systems in both hospitals, irradiance and radiance measurements were done according to the standard UNE-EN 62471. A Graphical User Interface (GUI) was developed for the spectral images processing and corresponding analysis, allowing spectral and colorimetric features to be computed in terms of reflectance, absorbance and colour parameters. Furthermore, a segmentation algorithm was also implemented to extract the isolated information from the lesion. For all images calculated in terms of any of the parameters, conventional statistical descriptors were obtained. As a first approach to extracting textural information we also used the analysis of the statistical properties of the histogram. An inclusion criteria and a measurement protocol were established. From all lesions analysed, 620 were measured with the multispectral system, 572 of them had a clinical or histopathological diagnosis, and 502 could be properly segmented. Therefore, 429 skin lesions were finally included in the study: 290 nevi, 95 melanomas and 44 basal cell carcinomas. A classification algorithm was developed in order to decide whether the lesions were malignant (melanomas and basal cell carcinomas) or not (nevi), splitting previously the data into training and validations set of the same size. 15 parameters from 1309 were found to be not redundant providing a 91.3% of sensitivity and 54.5% of specificity. Accordingly, the addition of textural information was shown to be useful for the diagnosis of malignant lesions than the sole use of averaged spectral and colour information. The same steps were carried out for the 3D imaging system also included in the multiphotonic platform. In this case, 3 parameters were found to be useful for the classification providing values of 55.6% and 83.7% of sensitivity and specificity, respectively. Finally, the combination of both system was also tested as a first attempt to improve the detection of melanomas, providing 100% and 72.2% of sensitivity and specificity, respectively. However, the conclusions reached in this case should be taken with caution due to the limited number of lesions.La incidència del càncer de pell a Europa, Estats Units i Austràlia ha anat augmentant ràpidament. Representa un de cada tres càncers a tot el món i una persona té 1:25 oportunitats de desenvolupar un melanoma, la forma més agressiva. Actualment, la inspecció visual amb un dermoscopi seguida d'un examen histològic és l'estàndard utilitzat pels metges a l'hora de diagnosticar-lo. La dermoscòpia requereix una formació considerable per interpretar el que es veu i depèn de les impressions subjectives dels clínics. En conseqüència, es realitzen una gran quantitat de procediments quirúrgics innecessaris. Per aquest motiu, en aquesta tesi s'ha desenvolupat una sistema d'imatge espectral per millorar el diagnòstic del càncer de pell. Aquest treball s'ha realitzat dins el marc del projecte Europeu DIAGNOPTICS ¿Diagnosis del càncer de pell utilitzant òptica?, el qual ha posat a punt un servei hospitalari basat en un plataforma multifotònica que combina quatre tècniques òptiques innovadores: sistemes d'imatge multiespectral 3D, interferometria de retroalimentació i microscòpia confocal. El sistema portàtil desenvolupat inclou una càmera monocromàtica CCD, un objectiu i una font de llum formada per 32 díodes emissors de llum (LED) amb 8 bandes espectrals diferents que emeten des de 400 nm fins a 1000 nm. S'ha preparat un programa d'adquisició per controlar tots els components del sistema així com una versió més simple del mateix pels metges. Per tal d'assegurar el bon funcionament del sistema, es van analitzar els canvis temporals en l'emissió dels LEDs així com la seva uniformitat d'emissió, la resposta lineal de la càmera per cada longitud d'ona i la repetibilitat del sistema pel què fa a l'adquisició d'imatges. Per tal d'obtenir l'aprovació del Comitè Ètic i poder realitzar l'estudi clínic en els hospitals, es van dur a terme mesures d'irradiància i radiància d'acord amb la norma UNE-EN 62471. També es va implementar una interfície gràfica d'usuari (GUI) per al processament de les imatges espectrals i la seva corresponent anàlisi. Aquest algorisme permet calcular paràmetres espectrals i colorimètrics de la pell en termes de reflectància, absorbància i d'altres basats en el color. A més, inclús es va desenvolupar un algorisme de segmentació per extreure informació aïllada de cada lesió. Per a totes les imatges calculades en termes de qualsevol paràmetre, es van obtenir descriptors estadístics convencionals i també es van utilitzar propietats estadístiques dels histogrames com una primera aproximació d'extreure informació de textura de la lesió. Finalment, es van establir els criteris d'inclusió i un protocol de mesura. De totes les lesions analitzades, se'n van mesurar 620, de les quals 572 tenien un diagnòstic clínic o histopatològic; 502 es van poder segmentar adequadament. D'aquesta manera es van incloure 429 lesions cutànies a l'estudi: 290 nevus, 95 melanomes i 44 carcinomes de cèl·lules basals. Es va desenvolupar un algorisme de classificació per determinar si les lesions eren malignes (melanomes i carcinomes de cèl·lules basals) o no (nevus), dividint prèviament les dades en un grup d'entrenament i un altre de validació de la mateixa mida. Es va observar que 15 del 1309 paràmetres proporcionaven informació rellevant per a la classificació, obtenint una sensibilitat del 91,3% i una especificitat del 54,5%. Així doncs, es demostra que la incorporació d'informació de textura és molt útil per al diagnòstic del càncer de pell més enllà de la informació espectral i de color. Aquests mateixos passos es van seguir pel sistema 3D també inclòs en la plataforma multifotònica, tot i que en aquest cas el número de lesions de què es disposava era més limitat. En aquest cas, es van seleccionar 3 paràmetres i es va obtenir una sensibilitat del 55,6% i una especificitat del 83,7%. Finalment, amb la combinació d'ambdós sistemes la sensibilitat obtinguda va ser de100% i l'especificitat del 72,2%

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

An Investigation of the Diagnostic Potential of Autofluorescence Lifetime Spectroscopy and Imaging for Label-Free Contrast of Disease

The work presented in this thesis aimed to study the application of fluorescence lifetime spectroscopy (FLS) and fluorescence lifetime imaging microscopy (FLIM) to investigate their potential for diagnostic contrast of diseased tissue with a particular emphasis on autofluorescence (AF) measurements of gastrointestinal (GI) disease. Initially, an ex vivo study utilising confocal FLIM was undertaken with 420 nm excitation to characterise the fluorescence lifetime (FL) images obtained from 71 GI samples from 35 patients. A significant decrease in FL was observed between normal colon and polyps (p = 0.024), and normal colon and inflammatory bowel disease (IBD) (p = 0.015). Confocal FLIM was also performed on 23 bladder samples. A longer, although not significant, FL for cancer was observed, in paired specimens (n = 5) instilled with a photosensitizer. The first in vivo study was a clinical investigation of skin cancer using a fibre-optic FL spectrofluorometer and involved the interrogation of 27 lesions from 25 patients. A significant decrease in the FL of basal cell carcinomas compared to healthy tissue was observed (p = 0.002) with 445 nm excitation. A novel clinically viable FLS fibre-optic probe was then applied ex vivo to measure 60 samples collected from 23 patients. In a paired analysis of neoplastic polyps and normal colon obtained from the same region of the colon in the same patient (n = 12), a significant decrease in FL was observed (p = 0.021) with 435 nm excitation. In contrast, with 375 nm excitation, the mean FL of IBD specimens (n = 4) was found to be longer than that of normal tissue, although not statistically significant. Finally, the FLS system was applied in vivo in 17 patients, with initial data indicating that 435 nm excitation results in AF lifetimes that are broadly consistent with ex vivo studies, although no diagnostically significant differences were observed in the signals obtained in vivo.Open Acces

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

Confocal Raman microscopy in skin drug delivery research

In drug research, growing scientific and regulatory demands increase the need for novel analytical methods. Especially in skin drug delivery research, advanced analytical techniques are urgently needed. The ability of contactless, label-free and chemically selective detection and quantification of substances predestines confocal Raman microscopy to fill this scientific gap. In this work, we implement confocal Raman microscopy in skin drug delivery research regarding reevaluation of existing in vitro approaches and acquisition of quantitative substance depth profiles in skin tissue. At first, we use Raman imaging to verify the suitability of porcine ears as in vitro model for follicular uptake. Furthermore, we investigate Raman signal attenuation as major drawback for quantitative depth profiling via the development of an artificial skin surrogate and evaluate the variability of Raman data acquired from different human skin donors. Moreover, we perform a systematic proof-of-concept study highlighting the possibility of quantitative depth profiling inside skin. Finally, we present freeze-drying as customized sample preparation technique to simplify and standardize quantitative depth profiling. Overall, the key parameters necessary for a standardized implementation of confocal Raman microscopy are understood and we elucidate crucial interdependences. This newly gained knowledge will help to advance current analytics in skin drug delivery research beyond the state of the-art.In der Arzneimittelforschung steigern wachsende wissenschaftliche und regulatorische Anforderungen den Bedarf nach neuen analytischen Methoden. Besonders in der dermalen Galenik sind die Ansprüche an die Analytik gestiegen. Durch die Fähigkeit zur kontaktfreien und chemisch selektiven Detektion und Quantifizierung von Substanzen hat konfokale Raman Mikroskopie das Potenzial diese Anforderungen zu erfüllen. In dieser Arbeit implementieren wir Raman Mikroskopie für die Neubewertung existierender in vitro Methoden und die Aufnahme quantitativer Tiefenprofile in Haut. Wir nutzen bildgebende Raman Mikroskopie um die Eignung von Schweineohren als in vitro Modell für follikulare Aufnahme zu bestätigen. Zudem bestimmen wir Raman-Signaldämpfung, das Haupthindernis bei der Erfassung von quantitativen Tiefenprofilen und bewerten die Variabilität von Raman-Spektren verschiedener menschlicher Hautproben. Ebenso zeigen wir in einer systematischen Machbarkeitsstudie erste quantitative Raman-Messungen in Haut und stellen Gefriertrocknung als maßgeschneiderte Probenaufbereitung für die vereinfachte und standardisierte Erfassung quantitativer Tiefenprofile vor. Insgesamt werden die entscheidenden Parameter für eine standardisierte Anwendung von Raman Mikroskopie verstanden und wichtige Zusammenhänge aufgeklärt. Auf dieser Grundlage kann sich konfokale Raman Mikroskopie durch weiter vereinfachte Messroutinen und spezialisierte Instrumente zu einer Standardmethode in der dermalen Arzneimittelforschung weiterentwickeln

Development of reflectance imaging methodologies to investigate super-paramagnetic iron oxide nanoparticles

Engineered nanoparticles, such as super paramagnetic iron oxide nanoparticles (SPIONs) offer significant benefits for the development of various diagnostic and therapeutic strategies. Limitations of existing imaging methodologies in the study of NPs, such as the effects of fluorescent labelling and diffraction limited resolution, and the advantages that visualization of spatial localization can offer in studies, increases the demand for new and optimized imaging routines. Reflectance Confocal Microscopy (RCM) methods were optimized and Reflectance Structured Illumination Microscopy (R-SIM) was introduced, offering a two fold increase in resolution - particularly advantageous for NP quantification and localization studies. Analysis routines were developed to enable the automated quantification of NP presence within cells via the different methodologies. Correlative procedures were also established for imaging the same sample with different reflectance methods and TEM, maximizing the information attainable from a single sample and allowing comparisons between the techniques for specific applications. These aforementioned optimized techniques were then applied to the determination of NP uptake and trafficking in cancer cell lines, and, in combination with siRNA, to ascertain proteins that are involved in the uptake process. Studies were also performed to model the degradative process of SPIONs within cellular compartments. This thesis thus provided several important tools for the future assessment of the efficacy and safety of NPs for clinical use, enabling quantitative analysis of uptake route, sub-cellular localization and NP intracellular fate

Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations

Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations. Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis. Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications