Development of a handheld fiber-optic probe-based raman imaging instrumentation: raman chemlighter

Raman systems based on handheld fiber-optic probes offer advantages in terms of smaller sizes and easier access to the measurement sites, which are favorable for biomedical and clinical applications in the complex environment. However, there are several common drawbacks of applying probes for many applications: (1) The fixed working distance requires the user to maintain a certain working distance to acquire higher Raman signals; (2) The single-point-measurement ability restricts realizing a mapping or scanning procedure; (3) Lack of real-time data processing and a straightforward co-registering method to link the Raman information with the respective measurement position. The thesis proposed and experimentally demonstrated various approaches to overcome these drawbacks. A handheld fiber-optic Raman probe with an autofocus unit was presented to overcome the problem arising from using fixed-focus lenses, by using a liquid lens as the objective lens, which allows dynamical adjustment of the focal length of the probe. An implementation of a computer vision-based positional tracking to co-register the regular Raman spectroscopic measurements with the spatial location enables fast recording of a Raman image from a large tissue sample by combining positional tracking of the laser spot through brightfield images. The visualization of the Raman image has been extended to augmented and mixed reality and combined with a 3D reconstruction method and projector-based visualization to offer an intuitive and easily understandable way of presenting the Raman image. All these advances are substantial and highly beneficial to further drive the clinical translation of Raman spectroscopy as potential image-guided instrumentation

Tunable lenses: Dynamic characterization and fine-tuned control for high-speed applications

Tunable lenses are becoming ubiquitous, in applications including microscopy, optical coherence tomography, computer vision, quality control, and presbyopic corrections. Many applications require an accurate control of the optical power of the lens in response to a time-dependent input waveform. We present a fast focimeter (3.8 KHz) to characterize the dynamic response of tunable lenses, which was demonstrated on different lens models. We found that the temporal response is repetitive and linear, which allowed the development of a robust compensation strategy based on the optimization of the input wave, using a linear time-invariant model. To our knowledge, this work presents the first procedure for a direct characterization of the transient response of tunable lenses and for compensation of their temporal distortions, and broadens the potential of tunable lenses also in high-speed applicationsVA and EL acknowledge financial support from Comunidad de Madrid and Marie Curie Action of the European Union FP7/2007-2013 COFUND 291820; XB from Comunidad de Madrid Doctorado Industrial IND2017/BMD-7670; EL from Spanish Government Ramon y Cajal Program RyC-2016-21125; EG from Spanish Government Torres-Quevedo Program PTQ-15-07432; LS from EU H2020 SME Innovation Associate GA-739882; EG from EIT Health; SM from ERC Grant Agreement ERC-2011-AdC 294099 and Spanish Government Grants FIS2014-56643-R; SM and CD from Spanish Government Grant FIS2017-84753-R; and CD from DTS16-0012

Modern Applications in Optics and Photonics: From Sensing and Analytics to Communication

Optics and photonics are among the key technologies of the 21st century, and offer potential for novel applications in areas such as sensing and spectroscopy, analytics, monitoring, biomedical imaging/diagnostics, and optical communication technology. The high degree of control over light fields, together with the capabilities of modern processing and integration technology, enables new optical measurement systems with enhanced functionality and sensitivity. They are attractive for a range of applications that were previously inaccessible. This Special Issue aims to provide an overview of some of the most advanced application areas in optics and photonics and indicate the broad potential for the future

New algorithms for the analysis of live-cell images acquired in phase contrast microscopy

La détection et la caractérisation automatisée des cellules constituent un enjeu important dans de nombreux domaines de recherche tels que la cicatrisation, le développement de l'embryon et des cellules souches, l’immunologie, l’oncologie, l'ingénierie tissulaire et la découverte de nouveaux médicaments. Étudier le comportement cellulaire in vitro par imagerie des cellules vivantes et par le criblage à haut débit implique des milliers d'images et de vastes quantités de données. Des outils d'analyse automatisés reposant sur la vision numérique et les méthodes non-intrusives telles que la microscopie à contraste de phase (PCM) sont nécessaires. Comme les images PCM sont difficiles à analyser en raison du halo lumineux entourant les cellules et de la difficulté à distinguer les cellules individuelles, le but de ce projet était de développer des algorithmes de traitement d'image PCM dans Matlab® afin d’en tirer de l’information reliée à la morphologie cellulaire de manière automatisée. Pour développer ces algorithmes, des séries d’images de myoblastes acquises en PCM ont été générées, en faisant croître les cellules dans un milieu avec sérum bovin (SSM) ou dans un milieu sans sérum (SFM) sur plusieurs passages. La surface recouverte par les cellules a été estimée en utilisant un filtre de plage de valeurs, un seuil et une taille minimale de coupe afin d'examiner la cinétique de croissance cellulaire. Les résultats ont montré que les cellules avaient des taux de croissance similaires pour les deux milieux de culture, mais que celui-ci diminue de façon linéaire avec le nombre de passages. La méthode de transformée par ondelette continue combinée à l’analyse d'image multivariée (UWT-MIA) a été élaborée afin d’estimer la distribution de caractéristiques morphologiques des cellules (axe majeur, axe mineur, orientation et rondeur). Une analyse multivariée réalisée sur l’ensemble de la base de données (environ 1 million d’images PCM) a montré d'une manière quantitative que les myoblastes cultivés dans le milieu SFM étaient plus allongés et plus petits que ceux cultivés dans le milieu SSM. Les algorithmes développés grâce à ce projet pourraient être utilisés sur d'autres phénotypes cellulaires pour des applications de criblage à haut débit et de contrôle de cultures cellulaires.Automated cell detection and characterization is important in many research fields such as wound healing, embryo development, immune system studies, cancer research, parasite spreading, tissue engineering, stem cell research and drug research and testing. Studying in vitro cellular behavior via live-cell imaging and high-throughput screening involves thousands of images and vast amounts of data, and automated analysis tools relying on machine vision methods and non-intrusive methods such as phase contrast microscopy (PCM) are a necessity. However, there are still some challenges to overcome, since PCM images are difficult to analyze because of the bright halo surrounding the cells and blurry cell-cell boundaries when they are touching. The goal of this project was to develop image processing algorithms to analyze PCM images in an automated fashion, capable of processing large datasets of images to extract information related to cellular viability and morphology. To develop these algorithms, a large dataset of myoblasts images acquired in live-cell imaging (in PCM) was created, growing the cells in either a serum-supplemented (SSM) or a serum-free (SFM) medium over several passages. As a result, algorithms capable of computing the cell-covered surface and cellular morphological features were programmed in Matlab®. The cell-covered surface was estimated using a range filter, a threshold and a minimum cut size in order to look at the cellular growth kinetics. Results showed that the cells were growing at similar paces for both media, but their growth rate was decreasing linearly with passage number. The undecimated wavelet transform multivariate image analysis (UWT-MIA) method was developed, and was used to estimate cellular morphological features distributions (major axis, minor axis, orientation and roundness distributions) on a very large PCM image dataset using the Gabor continuous wavelet transform. Multivariate data analysis performed on the whole database (around 1 million PCM images) showed in a quantitative manner that myoblasts grown in SFM were more elongated and smaller than cells grown in SSM. The algorithms developed through this project could be used in the future on other cellular phenotypes for high-throughput screening and cell culture control applications

Serial laser lithography for efficient manufacture of universal microstructures

The technique of microstructuring revolutionises all classical fields of engineering like electronics, optics and mechanics. In order to manufacture a microstructure in large quantities and at a reasonable price, master elements or masks will be formed that can be duplicated in a highly efficient process. Further development in technology leads, on the one hand, to further reduction of possible dimensions of structures down to the range of sub-nano technology and, on the other hand, to the development of more flexible systems in using more reasonably priced technologies for the structuring in the classical micrometre range, which in turn opens a much larger field of use. This study examines the use of serial laser lithography for efficient manufacture of universal microstructures. To facilitate this, a laser beam writer or so-called Laser Pattern Generator (LPG) was developed and described here as well as in a previous work[Samu96a]. The laser beam writer uses a precise positioning system for the movement of a substrate for material processing using a focussed laser beam. This system permits the production of structures with dimensions down to 0.5 μm which can be used in several application fields. This was systematically analysed for optimisation of the production process. Based on the achieved results, a computer-aided simulation system for process parameter determination and optimisation was developed that may be used in order to minimise the experimental effort in LPG manufacturing. The total production process and the individual optimising steps are illustrated by the manufacture of different microstructures. Because of the high reproducibility in manufacturing different structure types and, compared with other manufacturing methods, the low equipment and manufacturing effort, serial laser lithography is an efficient process for the microstructuring of universal microstructures down to the dimensions in the micrometre range

High Speed Confocal 3D Profilometer: Design, Development, Experimental Results

Ph.DDOCTOR OF PHILOSOPH

Small Particle Transport in Fibrin Gels and High Throughput Clot Characterization

The formation, function, and lysis of blood clots is largely governed by the transport of nano- and micro-scale particles. Yet there is not fully established physics that relates clot structure to transport phenomena such as fluid permeation and particle diffusion. This dissertation explores small particle transport in fibrin. I report on the size-dependence of particle mobility in fibrin, and discuss the implications of these results for fibrinolytic drug design. I measure the relationship between fibrin permeability and diffusion of 0.2--2.8 micron particles in fibrin gels, then determine the time and length scales at which small particle diffusion is directly related to bulk gel permeability. This result implies that one could develop a high throughput clot characterization assay that provides more detail than turbidity, the predominant high throughput measurement. I also present my work designing and developing systems to perform these and other experiments in high throughput, which include novel technologies for optical microscopy and magnetic force application.Doctor of Philosoph

Microcantilever biosensors

The cross-sensitivity of microcantilever sensors presents a major obstacle in the development of a commercially viable microcantilever biosensor for point of care testing. This thesis concerns electrothermally actuated bi-material microcantilevers with piezoresistive read out, developed for use as a blood coagulometer. Thermal properties of the sensor environment including the heat capacity and thermal conductivity affect the ‘thermal profile’ onto which the higher frequency mechanical signal is superimposed. In addition, polymer microcantilevers are known to have cross-sensitivity to relative humidity due to moisture absorption in the beam. However it is not known whether any of these cross sensitivities have a significant impact on performance of the sensor during pulsed mode operation or following immersion into liquid. When analysing patient blood samples, any change in signal that is not caused by the change in blood viscosity during clotting could lead to a false result and consequently an incorrect dose of anticoagulants may be taken by the patient. In order to address these issues three aspects of the operation of polymer bi-material strip cantilevers has been researched and investigated: relative humidity; viscosity/density, and thermal conductivity of a liquid environment. The relative humidity was not found to affect the resonant frequency of a microcantilever operated in air, or to affect the ability of the cantilever to measure clot times. However, a decrease in deflection with increasing relative humidity of the SmartStrip microcantilever beams is observed at 1.1 ± 0.4 μm per 1% RH, and is constant with temperature over the range 10 – 37 °C, which is an issue that should be considered in quality control. In this study, the SmartStrip was shown to have viscosity sensitivity of 2 cP within the range 0.7 – 15.2 cP, and it was also shown that the influence of inertial effects is negligible in comparison to the viscosity. To investigate cross-sensitivity to the thermal properties of the environment, the first demonstration of a cantilever designed specifically to observe the thermal background is presented. Characterisation experiments showed that the piezoresistive component of the signal was minimised to -0.8% ± 0.2% of the total signal by repositioning the read out tracks onto the neutral axis of the beam. Characterisations of the signal in a range of silicone oils with different thermal conductivities gave a resolution to thermal conductivity of 0.3 Wm-1K-1 and resulted in a suggestion for design improvements in the sensor: the time taken for the thermal background signal to reach a maximum can be increased by increasing the distance between the heater and sensor, thus lessening the impact of the thermal crosstalk within the cantilever beam. A preliminary investigation into thermal properties of clotting blood plasma showed that the sensor can distinguish the change between fresh and clotted plasma

Development of a miniaturized microscope for depth-scanning imaging at subcellular resolution in freely behaving animals

Le fonctionnement du cerveau humain est fascinant. En seulement quelques millisecondes, des milliards de neurones synchronisés perçoivent, traitent et redirigent les informations permettant le contrôle de notre corps, de nos sentiments et de nos pensées. Malheureusement, notre compréhension du cerveau reste limitée et de multiples questions physiologiques demeurent. Comment sont exactement reliés le fonctionnement neuronal et le comportement humain ? L’imagerie de l’activité neuronale au moyen de systèmes miniatures est l’une des voies les plus prometteuses permettant d’étudier le cerveau des animaux se déplaçant librement. Cependant, le développement de ces outils n’est pas évident et de multiples compromis techniques doivent être faits pour arriver à des systèmes suffisamment petits et légers. Les outils actuels ont donc souvent des limitations concernant leurs caractéristiques physiques et optiques. L’un des problèmes majeur est le manque d’une lentille miniature électriquement réglable et à faible consommation d’énergie permettant l’imagerie avec un balayage en profondeur. Dans cette thèse, nous proposons un nouveau type de dispositif d’imagerie miniature qui présente de multiples avantages mécaniques, électriques et optiques par rapport aux systèmes existants. Le faible poids, la petite dimension, la capacité de moduler électriquement la distance focale à l’aide d’une lentille à cristaux liquides (CL) et la capacité d’imager des structures fines sont au cœur des innovations proposées. Dans un premier temps, nous présenterons nos travaux (théoriques et expérimentaux) de conception, assemblage et optimisation de la lentille à CL accordable (TLCL, pour tunable liquid crystal lens). Deuxièmement, nous présenterons la preuve de concept macroscopique du couplage optique entre la TLCL et la lentille à gradient d’indice (GRIN, pour gradient index) en forme d’une tige. Utilisant le même système, nous démontrerons la capacité de balayage en profondeur dans le cerveau des animaux anesthésiés. Troisièmement, nous montrerons un dispositif d’imagerie (2D) miniature avec de nouvelles caractéristiques mécaniques et optiques permettant d’imager de fines structures neuronales dans des tranches de tissus cérébraux fixes. Enfin, nous présenterons le dispositif miniaturisé, avec une TLCL intégrée. Grâce à notre système, nous obtenons ≈ 100 µm d’ajustement électrique de la profondeur d’imagerie qui permet d’enregistrer l’activité de fines structures neuronales lors des différents comportements (toilettage, marche, etc.) de la souris.The functioning of the human brain is fascinating. In only a few milliseconds, billions of finely tuned and synchronized neurons perceive, process and exit the information that drives our body, our feelings and our thoughts. Unfortunately, our understating of the brain is limited and multiple physiological questions remain. How exactly are related neural functioning and human behavior ? The imaging of the neuronal activity by means of miniaturized systems is one of the most promising avenues allowing to study the brain of the freely moving subjects. However, the development of these tools is not obvious and multiple technical trade-offs must be made to build a system that is sufficiently small and light. Therefore, the available tools have different limitations regarding their physical and optical characteristics. One of the major problems is the lack of an electrically adjustable and energy-efficient miniature lens allowing to scan in depth. In this thesis, we propose a new type of miniature imaging device that has multiple mechanical, electrical and optical advantages over existing systems. The low weight, the small size, the ability to electrically modulate the focal distance using a liquid crystal (LC) lens and the ability to image fine structures are among the proposed innovations. First, we present our work (theoretical and experimental) of design, assembling and optimization of the tunable LC lens (TLCL). Second, we present the macroscopic proof-of-concept optical coupling between the TLCL and the gradient index lens (GRIN) in the form of a rod. Using the same system, we demonstrate the depth scanning ability in the brain of anaesthetized animals. Third, we show a miniature (2D) imaging device with new mechanical and optical features allowing to image fine neural structures in fixed brain tissue slices. Finally, we present a state-of-the-art miniaturized device with an integrated TLCL. Using our system, we obtain a ≈ 100 µm electrical depth adjustment that allows to record the activity of fine neuronal structures during the various behaviours (grooming, walking, etc.) of the mouse