The photothermal effect is a way in which chemical contrast can be measured as an optical pathlength or phase change. When a chemical species in a sample absorbs optical energy at a particular wavelength, this absorption raises the temperature at these points in the sample via the photothermal effect. This temperature change changes the local refractive index in the sample. Quantitative phase imaging is an interferometric technique for measuring the optical pathlength of sample features. Quantitative phase imaging is capable of detecting the photothermally-induced refractive index change, and is thus a powerful method for performing photothermal imaging. In this work, a thermal wave model is derived from Fourier's law of conduction in conjunction with a medium's heat capacity to derive the diffusion of temperature in a medium. This diffusion theory is transformed to a thermal wave model by applying a temporally modulated thermal source. Analytical expressions for the temperature field surrounding such a modulated thermal source are derived in multiple dimensions. The thermal wave equation is also simulated using a custom finite difference numerical method, and the simulated results are compared to the theoretical expressions with good agreement. The experimental apparatus for inducing such a thermal point source in a medium of water is described using the quantitative phase imaging system of spectral modulation interferometry. The spectral modulation interferometry system is aligned with a visible light pumping laser in two configurations for point source measurement and cell imaging. Label-free chemical imaging is then performed by pumping a field of cellular samples with wide-field illumination, and the resulting photothermal signal is detected by temporal analysis of the optical pathlength changes, generating the two-dimensional photothermal image. The measured photothermal cell image is qualitatively compared to predicted photothermal image based on the application of the thermal wave model in the spatial frequency domain. The chemical specificity of this technique is also verified by simultaneously pumping absorbing and non-absorbing biological cells in the same field-of-view. / Generating image contrast is a fundamental challenge in optical microscopy. Samples of interest in optical microscopy typically do not have visible absorption contrast without modification. A method of contrast that could provide information about a sample's absorption at different optical wavelengths would be useful for characterizing a sample's chemical content. The photothermal effect is an effect in which the small absorption of light by microscopic samples can be detected as a temperature change. With quantitative phase imaging, this temperature change can be measured by detecting the change in optical density of a sample due to its increase in temperature. Thus, quantitative phase imaging can be used to detect the small absorption of light by microscopic samples and generate two-dimensional images with chemical contrast. This work describes the theory of how thermal energy produced by optical absorption diffuses through a sample immersed in water. A thermal wave model is derived theoretically and compared to a custom simulation of the thermal wave physics with strong agreement. This thermal theory is verified with the quantitative phase imaging system used in this work to characterize the photothermal imaging technique. The photothermal imaging method is then applied to cellular samples, which are pumped with green light. The photothermal image is then generated and compared qualitatively to the image predicted by the thermal theory. The chemical imaging ability of the technique is then demonstrated by simultaneous imaging of absorbing and non-absorbing cells.

Analyzing the structure of a single cell based on its refractive index (RI) distribution is a common and valued approach, because it does not require any artificial markers. The RI is an inherent structural marker that can be quantified in three dimensions with optical diffraction tomography (ODT), an inverse scattering technique. This work reviews the theory of ODT and its implementation with an emphasis on single-cell analysis, identifying the Rytov approximation as the most efficient descriptor for light propagation. The accuracy of the reconstruction method is verified with in silico data and imaging artifacts associated with the inverse scattering approach are addressed. Furthermore, an experimental ODT setup is presented that consists of a bright-field microscope, a phase-imaging camera, and an optical trap combined with a microfluidic chip. A novel image analysis pipeline is proposed that addresses image corrections and frame alignment of the recorded data prior to the RI reconstruction. In addition, for a rotational axis that is tilted with respect to the image plane, an improved reconstruction algorithm is introduced and applied to single, suspended cells in vitro, achieving sub-cellular resolution.

Wavefront sensing is a fundamental problem in applied optics. Wavefront sensors that work in a deterministic manner are of particular interest. Initialized with a unified theory for classical wavefront sensors, this dissertation discusses relevant properties of wavefront sensor designs. Based on which, a new wavefront sensor, termed Coded Wavefront Sensor, is proposed to leverage the advantages of the analysis, especially the lateral wavefront resolution. A prototype was built to demonstrate this new wavefront sensor. Given that, two specific applications are demonstrated: megapixel adaptive optics and simultaneous intensity and phase imaging. Combined with a spatial light modulator, a hardware deconvolution approach is demonstrated for computational cameras via a high resolution adaptive optics system. By simply switching the normal image sensor with the proposed one, as well as slight change of illumination, a bright field microscope can be configured to a simultaneous intensity and phase microscope. These show the broad application range of the proposed computational wavefront sensing approach. Lastly, this dissertation proposes the idea of differentiable optics for wavefront engineering and lens metrology. By making use of automatic differentiation, a physically-correct differentiable ray tracing engine is built, with its potentials being illustrated via several challenging applications in optical design and metrology.

La microscopie optique est une technique essentielle pour de nombreuses disciplines des sciences expérimentales qui nécessitent des résolutions sans cesse plus petites. Dans ce travail de thèse sont présentés plusieurs travaux pour l'amélioration de la résolution en microscopie de fluorescence et en microscopie tomographique par diffraction (MTD), une récente technique de microscopie de phase. Dans un premier temps, il est montré que déposer l'échantillon sur un miroir permet d'augmenter la résolution axiale en MTD et en microscopie confocale de fluorescence. En microscopie confocale, il faut pour cela mettre en forme le faisceau incident grâce à un modulateur spatial de lumière. En MTD, il suffit d'adapter le programme de reconstruction. La deuxième partie présente des algorithmes pour reconstruire des images haute résolution à partir de mesures en éclairement structuré avec de champs d'illumination inconnus, à la fois en microscopie de fluorescence (algorithme blind-SIM) et en MTD. En microscopie de fluorescence, ces algorithmes permettent de simplifier drastiquement les montages expérimentaux produisant l'éclairement structuré et en MTD, d'obtenir des images d'échantillons à fort indice. / Various fields of experimental science are constantly requiring smaller resolution for optical microscopy. In this thesis are presented several works for improving resolution in fluorescence microscopy and in Tomographic Diffraction Microscopy (TDM), an emerging phase microscopy technique. In the first part it is shown that one can improve the axial resolution in depositing the sample on a mirror. In confocal fluorescence microscopy, this is done by shaping the illumination beam with a Spatial Light Modulator. In TDM this is done by adapting the reconstruction method. Then algorithms are proposed for reconstructing high-resolution images from structured illumination measurements with unknown illumination fields, both in fluorescence imaging (blind-SIM algorithm) and in TDM. This allows a dramatical simplification of the experimental set-ups in fluorescence structured illumination and the image reconstruction of high optical index samples in TDM.

Analyzing the structure of a single cell based on its refractive index (RI) distribution is a common and valued approach, because it does not require any artificial markers. The RI is an inherent structural marker that can be quantified in three dimensions with optical diffraction tomography (ODT), an inverse scattering technique. This work reviews the theory of ODT and its implementation with an emphasis on single-cell analysis, identifying the Rytov approximation as the most efficient descriptor for light propagation. The accuracy of the reconstruction method is verified with in silico data and imaging artifacts associated with the inverse scattering approach are addressed. Furthermore, an experimental ODT setup is presented that consists of a bright-field microscope, a phase-imaging camera, and an optical trap combined with a microfluidic chip. A novel image analysis pipeline is proposed that addresses image corrections and frame alignment of the recorded data prior to the RI reconstruction. In addition, for a rotational axis that is tilted with respect to the image plane, an improved reconstruction algorithm is introduced and applied to single, suspended cells in vitro, achieving sub-cellular resolution.

The Fourier transform (spectrum) of a signal is a complex function and is characterized by the magnitude and phase spectra. Phase retrieval is the reconstruction of the phase spectrum from the measurements of the magnitude spectrum. Such problems are encountered in imaging modalities such as X-ray crystallography, frequency-domain optical coherence tomography (FDOCT), quantitative phase microscopy, digital holography, etc., where only the magnitudes of the wavefront are detected by the sensors. The phase retrieval problem is ill-posed in general, since an in nite number of signals can have the same magnitude spectrum. Typical phase retrieval techniques rely on certain prior knowledge about the signal, such as its support or sparsity, to reconstruct the signal. A classical result in phase retrieval is that minimum-phase signals have log-magnitude and phase spectra that satisfy the Hilbert integral equations, thus facilitating exact phase retrieval. In this thesis, we demonstrate that there exist larger classes of signals beyond minimum-phase signals, for which exact phase retrieval is possible. We generalize Hilbert integral equations to 2-D, and also introduce a variant that we call the composite Hilbert transform in the context of 2-D periodic signals. Our first extension pertains to a particular type of parametric modelling of 2-D signals. While 1-D minimum-phase signals have a parametric representation, in terms of poles and zeros, there exists no such 2-D counterpart. We introduce a new class of parametric 2-D signals that possess the exact phase retrieval property, that is, their magnitude spectrum completely characterizes the signal. Starting from the magnitude spectrum, a sequence of non-linear operations lead us to a sum-of-exponentials signal, from which the parameters are computed employing concepts from high-resolution spectral estimation such as the annihilating filter and algebraically coupled matrix-pencil methods. We demonstrate that, for this new class of signals, our method outperforms existing techniques even in the presence of noise. Our second extension is to continuous-domain signals that lie in a principal shift-invariant space spanned by a known basis. Such signals are characterized by the basis combining coefficients. These signals need not be minimum-phase, but certain conditions on the coefficients lead to exact phase retrieval of the continuous-domain signal. In particular, we introduce the concept of causal, delta dominant (CDD) sequences, and show that such signals are characterized by their magnitude spectra. This condition pertains to the time/spatial-domain description of the signal, in contrast to the minimum-phase condition, which is described in the spectral domain. We show that there exist CDD sequences that are not minimum-phase, and vice versa. However, finite-length CDD sequences are always minimum-phase. Our method reconstructs the signal from the magnitude spectrum up to ma-chine precision. We thus have a class of continuous-domain signals that are neither causal nor minimum phase, and yet allow for exact phase retrieval. The shift-invariant structure is applicable to modelling signals encountered in imaging modalities such as FDOCT. We next present an application of 2-D phase retrieval to continuous-domain CDD signals in the context of quantiative phase microscopy. We develop sufficient conditions on the interfering reference wave for exact phase retrieval from magnitude measurements. In particular, we show that when the reference wave is a plane wave with magnitude greater that the intensity of the object wave, and when the carrier frequency is larger than the band-width of the object wave, we can reconstruct the object wave exactly. We demonstrate high-resolution reconstruction of our method on USAF target images. Our final and perhaps the most unifying contribution is in developing Hilbert integral equations for 2-D first-quadrant signals and in introducing the notion of generalized minimum-phase signals for both 1-D and 2-D signals. For 2-D continuous-domain, first-quadrant signals, we establish partial Hilbert transform relations between the real and imaginary parts of the spectrum. In the context of 2-D discrete-domain signals, we show that the partial Hilbert transform does not suffice and introduce the notion of composite Hilbert transform and establish the integral equations. We then introduce four classes of signals (combinations of 1-D/2-D and continuous/discrete-domain) that we call generalized minimum-phase signals, which satisfy corresponding Hilbert integral equations between log-magnitude and phase spectra, hence facilitating exact phase retrieval. This class of generalized minimum-phase signals subsumes the well known class of minimum-phase signals. We further show that, akin to minimum-phase signals, these signals also have stable inverses, which are also generalized minimum-phase signals.

Cette thèse vise à améliorer la résolution en trois dimensions grâce à une technique récente d’imagerie : la microscopie tomographique diffractive (MTD). Son principe est d’éclairer l’objet successivement sous différents angles en lumière cohérente, de détecter le champ diffracté en phase et en amplitude, et de reconstruire la carte 3D de permittivité de l’objet par un algorithme d’inversion. La MTD s’est avérée capable de combiner plusieurs modalités utiles pour la microscopie sans marquage, telles que plein champ, champ sombre, à contraste de phase, confocale, ou encore la microscopie à synthèse d’ouverture 2D ou 3D. Toutes sont basées sur des approximations scalaires et linéaires, ce qui restreint leur domaine d’application pour restituer l’objet de manière quantitative. A l’aide d’une inversion numérique rigoureuse prenant en compte la polarisation du champ et le phénomène de diffusion multiple, nous sommes parvenus à reconstruire la carte 3D de permittivité d’objets avec une résolution de λ/4. Une amélioration supplémentaire la portant à λ/10 a été rendue possible par l’insertion d’information a priori sur l’objet dans l’algorithme d’inversion. Enfin, la résolution axiale est moins bonne du fait de l’asymétrie des schémas d’illumination et de détection dans les microscopes. Pour s’affranchir de cette limitation, une configuration de tomographie assistée par miroir a été implémentée et a mis en évidence un pouvoir de séparation axial meilleur que λ/2. Au final, la MTD s’est illustrée comme un outil de caractérisation puissant pour reconstruire en 3D les objets ainsi que leurs indices optiques, à des résolutions bien supérieures à celles des microscopes conventionnels. / This PhD thesis is devoted to the three-dimensional isotropic resolution improvement using optical tomographic diffraction microscopy (TDM), an emerging optical microscope technique. The principle is to illuminate the sample successively with various angles of coherent light, collect the complex (amplitude and phase) diffracted field and reconstruct the sample 3D permittivity map through an inversion algorithm. A single TDM measurement was shown to combine several popular microscopy techniques such as bright-field microscope, dark-field microscope, phase-contrast microscope, confocal microscope, 2D and 3D synthetic aperture microscopes. All rely on scalar and linear approximations that assume a linear link between the object and the field diffracted by it, which limit their applicability to retrieve the object quantitatively. Thanks to a rigorous numerical inversion of the TDM diffracted field data which takes into account the polarization of the field and the multiple scattering process, we were able to reconstruct the 3D permittivity map of the object with a λ/4 transverse resolution. A further improvement to λ/10 transverse resolution was achieved by providing a priori information about the sample to the non-linear inversion algorithm. Lastly, the poor axial resolution in microscopes is due to the fundamental asymmetry of illumination and detection. To overcome this, a mirror-assisted tomography configuration was implemented, and has demonstrated a sub-λ/2 axial resolution capability. As a result, TDM can be seen as a powerful tool to reconstruct objects in three-dimensions with their optical material properties at resolution far superior to conventional microscopes.

Le premier chapitre de cette thèse traite de l'importance des échelles temporelles et spatiales dans le contexte des systèmes vivants. J'y décris également les principaux composants de la réponse mécanique des cellules vivantes. Après ce chapitre introductif, le deuxième chapitre est dédié à la réponse mécanique des cellules évaluée avec l'AFM et en particulier, son aspect dynamique. Je présente d'abord l'analyse des courbes force-indentation, puis je propose une méthode alternative pour l'étude de la rhéologie cellulaire qui est basée sur l'excitation multifréquence du levier par bruit thermique. La DPM est l'objet du troisième chapitre où je revisite la méthode d'extraction de phase en utilisant la transformation en ondelette à deux dimensions. Ensuite je montre comment la DPM peut être utilisée pour caractériser les fluctuations temporelles et la morphologie de différents types de cellules du sang et de cellules adhérentes. Finalement, le chapitre quatre est un chapitre de conclusion où je fais une synthèse des résultats obtenus. Par exemple, je montre que, en comparaison avec des cellules saines, les cellules leucémiques subissent des changements morphologiques qui sont accompagnés par un comportement mécanique plus rigide et plus élastique. Cela indique que dans cet exemple la transformation cellulaire n'est pas seulement donnée par son cortex mais aussi par son cytosquelette et son couplage avec le noyau / In the first chapter of this thesis I discuss the importance of spatial and temporal scales in living systems, and I review the main components involved in the mechanical response of living cells. After this introductory chapter, the second one is dedicated to evaluating the mechanical response of single-cells with AFM, and in particular, its dynamical aspect. I present the analysis of force-indentation curves without any assumption on the linearity of the system, contrary to more typical analysis based on Sneddon’s or Hertz models. Then, I propose an alternative method to study the cell rheology based on the multi-frequency excitation of the cantilever by thermal noise. DPM is discussed on chapter three. I revisit the phase recovery method using the 2D wavelet transform, and I show how DPM can be used to characterize the temporal fluctuations and the morphology of different types of blood cells and adherent cells. Finally, chapter four is a conclusion chapter where I summarise our results by comparing healthy and pathological immature blood cells. For instance I show that, in comparison to healthy cells, leukaemic cells undergo morphological changes that are accompanied by a stiffer and more elastic behaviour. Altogether, our results indicate that this cell transformation involves the whole cytoskeleton and its coupling to the nucleus rather than simply the cell cortex

[ES] La tomografía por difracción surge para mejorar las técnicas de imagen al considerar la naturaleza ondulatoria de la luz. Mientras que los primeros sistemas de imagen médica se basaban únicamente en fuentes sin difracción, este enfoque consigue mejorar la reconstrucción del índice de refracción de los objetos, lo que permite, por ejemplo, el estudio de estructuras subcelulares. Del mismo modo, la demanda de redes de telecomunicaciones cada vez más rápidas y seguras ha propiciado la aparición de la fotónica. Hace dos décadas, la combinación de estos dos campos dio lugar a los primeros sistemas de tomografía por difracción óptica (ODT), los cuáles han evolucionado rápidamente durante este siglo. En esta tesis, presentamos dos nuevas aplicaciones de la ODT. La primera está relacionada con el concepto del microscopio tomográfico de fase (TPM), una versión de la ODT que permite el estudio de células aisladas, con muchas aplicaciones biomédicas, como el diagnóstico y la prognosis del cáncer. Sin embargo, los sistemas TPM actuales son caros, pesados y complejos. Para resolver estos problemas, proponemos el concepto de TPM en chip. Con este fin, diseñamos una hoja de ruta hacia el primer dispositivo tomográfico integrado en el marco de la tecnología lab-on-a-chip (LoC), y desarrollamos los primeros pasos para ello: 1) Hasta ahora, sólo se han utilizado detectores planos para obtener los mapas de índice de refracción de los objetos estudiados en TPM, basados en la detección del campo difractado hacia delante. Sin embargo, los principios físicos fundamentales indican que medir también el campo difractado hacia detrás debería mejorar la resolución de las imágenes. Además, un detector plano no es la configuración óptima para el TPM en chip. En esta línea, hemos explorado la posibilidad de usar detectores circulares en este escenario, como una técnica más adecuada para las configuraciones en chip, demostrando al mismo tiempo que este enfoque proporciona una mejor resolución que el lineal. 2) Proponemos un esquema de TPM en chip basado en el uso de nanoantenas dieléctricas como fuente de luz y píxeles detectores ODT, y caracterizamos experimentalmente su comportamiento mediante microscopía óptica de campo cercano. En cuanto a la segunda aplicación, estudiamos el potencial de la ODT como nuevo paradigma en la detección de capas de invisibilidad realistas, una de las aplicaciones más importantes de los metamateriales. Hasta ahora, el scattering cross section (SCS) se ha utilizado como modelo de referencia para diseñar y observar la eficacia de estos dispositivos para ocultar objetos. En nuestro estudio, demostramos que la ODT puede detectar las capas de invisibilidad prácticas con una sensibilidad superior a la que ofrece el SCS, incluso a las frecuencias de trabajo óptimas. Además, es posible obtener una imagen representativa del tamaño y la forma de la capa, revelando claramente su existencia. Finalmente, se discuten las conclusiones extraídas de los resultados obtenidos. Además, se detallan las futuras líneas de trabajo para abordar los retos que no se han completado en esta tesis doctoral. / [CA] La tomografia per difracció sorgeix per millorar les tècniques d'imatge anteriors en considerar la naturalesa ondulatòria de la llum. Mentre que els primers sistemes d'imatge mèdica es basaven únicament en fonts sense difracció, aquest enfocament aconsegueix millorar la reconstrucció de l'índex de refracció dels objectes, la qual cosa permet, per exemple, l'estudi d'estructures subcelulars. De la mateixa manera, la demanda de xarxes de telecomunicacions cada vegada més ràpides i segures ha propiciat l'aparició de la fotònica. Fa dues dècades, la combinació d'aquests dos camps va portar als primers sistemes de tomografia per difracció òptica (ODT), els quals han evolucionat ràpidament durant aquest segle. En aquesta tesi, presentem dues noves aplicacions de la ODT. La primera està relacionada amb el concepte del microscopi tomogràfic de fase (TPM), una versió de la ODT que permet l'estudi de cèl·lules aïllades, amb moltes aplicacions en biomedicina, com el diagnòstic i prognosi del càncer. No obstant això, els sistemes TPM actuals són cars, pesats i complexos. Per resoldre aquests problemes, proposem el concepte de TPM en xip. Per fer-ho, dissenyem un full de ruta cap al primer dispositiu tomogràfic integrat en el marc de la tecnologia lab-on-a-chip (LoC), i desenvolupem els primers passos a aquest efecte: 1) Fins ara, només s'han utilitzat detectors plans per a obtindre els mapes d'índex de refracció dels objectes estudiats en TPM, basats en la detecció del camp difractat cap avant. No obstant això, els principis físics fonamentals indiquen que mesurar també el camp difractat cap endarrere hauria de millorar la resolució de les imatges. A més, un detector pla no és la configuració òptima per al TPM en xip. En aquesta línia, hem explorat la possibilitat d'usar detectors circulars en aquest escenari, com una tècnica més adequada per a les configuracions en xip, demostrant al mateix temps que aquest enfocament proporciona una millor resolució que el lineal. 2) Proposem un esquema de TPM en xip basat en l'ús de nanoantenes dielèctriques com a font de llum i píxels detectors ODT, i caracteritzem experimentalment el seu comportament en camp pròxim mitjançant microscòpia òptica de camp pròxim. Pel que fa a la segona aplicació, estudiem el potencial de la ODT com a nou paradigma en la detecció de capes d'invisibilitat realistes, una de les aplicacions més importants dels metamaterials. Fins ara, el scattering cross section (SCS) s'ha utilitzat com a model de referència per a dissenyar i observar l'eficàcia d'aquests dispositius per a ocultar objectes. En el nostre estudi, vam demostrar que la ODT pot detectar les capes d'invisibilitat pràctiques amb una sensibilitat superior a la que ofereix el SCS, fins i tot a les freqüències de treball òptimes. A més, és possible obtindre una imatge representativa de la grandària i la forma de la capa, revelant clarament la seua existència. Finalment, es discuteixen les conclusions extretes dels resultats obtinguts i es detallen les futures línies de treball per a abordar els reptes que no s'han completat en aquesta tesi doctoral. / [EN] Diffraction Tomography arises to improve previous imaging techniques by considering the wave nature of light. Whereas the first medical imaging systems relied only on non-diffracting sources, this approach results in an enhanced reconstruction of the object's refractive index distribution, allowing, for example, the study of subcellular structures. Likewise, the demand for increasingly faster and secure telecommunication networks led to the advent of photonics. Two decades ago, the combination of these two fields gave rise to the first optical diffraction tomography (ODT) systems, which have rapidly evolved during this century. In this thesis, we present two novel applications of ODT. The first one is related to the concept of tomographic phase microscopy (TPM), a version of ODT that enables the study of isolated cells, with many applications in biomedicine, such as the diagnosis and prognosis of cancer. Nevertheless, current TPM systems are expensive, heavy, and cumbersome. To solve these issues we propose the concept of on-chip TPM. For this purpose, we design a roadmap towards the first integrated tomographic device in the frame of lab-on-a-chip (LoC) technology and develop the first steps to this end: 1) Until now, only flat detectors have been used to obtain the refractive index maps of the objects studied in TPM, based on the detection of the forward scattering. However, fundamental physical principles indicate that measuring also the backscattered field should improve the resolution of the images. Moreover, a flat detector is not the optimal configuration for on-chip TPM. In this vein, we have explored the possibility of using circular detectors in this scenario as a more suitable technique for on-chip configurations, demonstrating at the same time that this approach provides a better resolution than the linear one. 2) We propose a TPM on-chip scheme based on the use of dielectric nanoantennas as the ODT light source and detector pixels, and experimentally characterize their near-field behavior via scanning near-field optical microscopy. As for the second application, we study the potential of ODT as a new paradigm in the detection of realistic invisibility cloaks, one of the most important applications of metamaterials. Up to now, the scattering cross section (SCS) has been used as the gold standard to design and observe the effectiveness of these devices in hiding objects. In our study, we show that ODT can detect practical invisibility cloaks with a higher sensitivity than that offered by the SCS, even at the optimal working frequencies. Moreover, it is possible to obtain an image depicting the size and shape of the cloak, clearly revealing their existence. Finally, the conclusions drawn from the obtained results are discussed. In addition, future lines of action to address the challenges that have not been completed in this doctoral thesis are detailed. / Díaz Fernández, FJ. (2021). Novel Applications of Optical Diffraction Tomography: On-chip Microscopy and Detection of Invisibility Cloaks [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/180125