The dissertation presents theoretical and experimental studies on the physical origin of the signal in photothermal microscopy of single particles. This noninvasive optical far field microscopy scheme allows the imaging and detection of single absorbing nanoparticles. Based on a heat-induced pertur- bation in the refractive index in the embedding medium of the nanoscopic absorber, a corresponding probe beam modification is measured and quantified. The method is well established and has been applied since its first demonstration in 2002 to the imaging and characterization of various absorbing particle species, such as quantum dots, single molecules and nanoparticles of different shapes. The extensive theoretical developments presented in this thesis provide the first quantitative assess- ment of the signal and at the same time enlarge its phenomenology and thereby its potential. On the basis of several approximation schemes to the Maxwell equations, which fundamentally gov- ern the interaction of light with inhomogeneities, several complementing models are devised which describe the photothermal signal both qualitatively and quantitatively. In succession an interdepen- dent and self-consistent set of theoretical descriptions is given and allows important experimental consequences to be drawn. In consequence, the photothermal signal is shown to correspond to the action of a nanoscopic (thermal) lens, represented by the spherically symmetric refractive index pro- file n(r) which accompanies the thermal expansion of the absorber’s environment. The achieved quantification allows the direct measurement of absorption cross-sections of nanoparticles. Further, a qualitatively new phenomenology of the signal is unraveled and experimentally demonstrated. The separate roles of the probing and the heating beams in photothermal microscopy is dismantled and the influence of their relative alignment shown to allow for a controlled adjustment of the effective detection volume. For the first time, both positive and negative signals are demonstrated to occur and to be the characteristic signature of the lens-like action on the probe beam. The detection of the probe beam’s modification is also shown to sensitively depend on the aperture used in the detection chan- nel, and a signal optimization is shown to be feasible. Also, a generalization of the detectable signal via the use of a quadrant photodiode is achieved. Specifically, measuring the far field beam deflec- tion the result of the beam passing the lens off-center manifests in a laterally split detection volume. Hereby, finally each classical photothermal spectroscopic techniques has been shown to possess its microscopic counterpart. Central to the understanding of this generalized and new phenomenology is a scalar wave-optical model which draws an analogy between the scattering of a massive particle wave-packet by a Coulomb potential and the deflection of a focused beam by a photonic potential connected with the thermal lens. The significance of the findings is demonstrated by its methodological implications on photother- mal correlation spectroscopy in which the diffusion dynamics of absorbing colloidal particles can be studied. The unique split focal detection volumes are shown to allow the sensitive measurement of a deterministic velocity field. Finally, the method is supplemented by a newly introduced sta- tistical analysis method which is capable of characterizing samples containing a heterogeneous size distribution.

For the proper function of the Fluorescence olographic microscope, it is necessary to adjust all the optical components of the microscope. Furthermore, the precise adjustment is the very critical condition for proper imaging of the Coherence-controlled holographic microscope. Therefore, it is necessary to create a sight collimator for these microscopes for their adjustment. The fluorescence holographic microscope is based on an interference and holographic principles, whose history is mentioned in the theoretical part of the thesis. The existing state of the art of laser sight collimators and their use in practice is also mentioned. The optical and mechanical design of the laser sight collimator and its realization are described in the next part of the thesis. The software for detecting the black sight cross was created for the use of the laser sight collimator in practice. The software is necessary to evaluate the correctness of the alignment of the adjusted microscope. The descriptions of the adjustment procedures for the laser sight collimator and for the Fluorescence holographic microscope are mentioned in the last part of the thesis. These procedures are necessary for proper manipulation and use with the proposed laser sight collimator.

La microscopie par cohérence optique (OCM) est une technique d'imagerie tomographique basée sur l'interférométrie en lumière blanche permettant d'imager les milieux biologiques à l'échelle microscopique. L'OCM est une méthode particulièrement adaptée à l'imagerie dermatologique, en particulier pour le diagnostic du cancer de la peau, car elle permet d'obtenir des images similaires aux images histologiques sans nécessiter d'effectuer de biopsie.Ces travaux de thèse portent sur le développement de la microscopie par cohérence optique pour l'imagerie de la peau, dans le but de fournir au dermatologue un outil d'imagerie compact, adapté à l'imagerie dermatologique in vivo, et permettant d'obtenir des images à la fois structurelles et fonctionnelles.Un dispositif de microscopie par cohérence optique plein champ (FF-OCM) compact, à éclairage par LED blanche, a tout d'abord été développé, permettant d'obtenir des images tomographiques à très haute résolution (0.7 μm × 1.8 μm) jusqu’à ∼200 μm de profondeur dans la peau. En utilisant une LED de haute puissance, des images de peau in vivo ont pu être obtenues.A partir de ce dispositif de FF-OCM, des méthodes d'imagerie fonctionnelle permettant de cartographier les écoulements sanguins (angiographie) ont été mises en oeuvre. Quatre méthodes, basées sur une analyse du signal interférométrique (temporelle ou fréquentielle), d'images de phase ou d'images d'amplitude ont permis d'imager de l'intralipide s'écoulant dans un modèle de capillaire sanguin.L'imagerie fonctionnelle polarimétrique a aussi été explorée en FF-OCM. Une optimisation du contraste des images polarimétriques a été obtenue en modifiant les composants polarisants d'un montage conventionnel de FF-OCM polarimétrique en fonction de l'échantillon imagé. Cette méthode a été testée sur un échantillon polarisant simple.Finalement, une nouvelle méthode d'OCM, la microscopie par cohérence optique confocale à éclairage « ligne » (LC-OCM) a été étudiée, dans le but de développer un système permettant d'imager la peau in vivo, avec une plus grande profondeur de pénétration dans les tissus que la FF-OCM. Ce système, combinant un filtrage interférométrique et un filtrage confocal, a permis d'obtenir des images de peau in vivo en coupe verticale et en coupe en face, avec une résolution spatiale similaire à celle de la FF-OCM, mais à une profondeur supérieure atteignant 300 μm. / Optical coherence microscopy (OCM) is a technique for tomographic imaging based on white light interferometry, making it possible to image biological media with micrometer-scale spatial resolution. OCM is particularly well-suited to dermatological imaging, especially skin cancer diagnosis, since it provides images that are similar to histological images without the need for biopsy.This PhD thesis focuses on the development of OCM for skin imaging, with the aim of providing a compact, in vivo imaging tool for the dermatologist, capable of acquiring structural and functional images of the skin.A compact, full-field OCM (FF-OCM) system illuminated by a white LED was first developed, making it possible to obtain tomographic images at an ultra-high resolution (0.7 μm × 1.8 μm), up to ∼200 μm in depth within the skin. Using a high power LED, in vivo skin images could be obtained.Using this FF-OCM setup, functional imaging methods for blood flow mapping (angiography) were implemented. Four methods, based on temporal or frequency analysis of the interferometric signal, phase images or amplitude images, have been shown to be able to image intralipid flow within a model blood capillary.Functional polarimetric imaging has also been explored in FF-OCM. Contrast optimization in polarimetric images has been obtained by modifying the polarizing components of the conventional polarization sensitive FF-OCM setup depending on the sample to be imaged. This method has been tested on a simple polarizing sample.Finally, a new OCM method, line-field confocal OCM (LC-OCM), has been studied. The goal here was to develop a system capable of imaging the skin in vivo, with a tissue penetration depth greater than what is possible for FF-OCM. This system, which combines interferometric filtering and confocal filtering, makes it possible to obtain in vivo skin images in vertical and en face slices, with a spatial resolution similar to that of FF-OCM, but with a greater penetration depth of 300 μm.

La microscopie interférométrique est une méthode de mesure qui repose sur l’acquisition et le traitement du signal issu de l’interaction de deux ondes, dites ondes « objet » et de « référence ». Ces ondes proviennent des réflexions de la lumière sur un miroir de référence et sur l’échantillon étudié. Bien qu’étant généralement utilisées pour les analyses topographiques ou tomographiques d’un échantillon, les données interférométriques peuvent être exploitées pour réaliser des caractérisations spectrales locales résolues dans les trois directions de l’espace. Dans ce projet, nous avons étudié les performances de cette technique ainsi que ses limitations lorsque l’échantillon se complexifie (dégradation du signal d’interférences). L’analyse a été appliquée à des matériaux réfléchissants pour des mesures en surface puis à des couches transparentes et diffusantes pour aller sonder le milieu en profondeur et extraire la réponse spectrale individuelle de structures localisées dans ce milieu. / White light interference microscopy is a measurement method based on the acquisition and processing of the signal coming from the interaction between two wave fronts, known as the “object” and “reference” wave-fronts. These waves come from the reflection of the light on a reference mirror and the sample studied. Usually used for topographic or tomographic analysis of a sample, the interferometric data can be exploited for spectroscopic purposes. The resulting spectral characterizations are spatially resolved in the three directions of space. In this project, we have studied the performance of this technique, as well as the associated limitations when the sample becomes more complex (degradation of the interferometric signal). The analysis has been first applied to reflective materials for surface measurements and subsequently to transparent and scattering layers for probing within the depth of the medium and then extracting the individual spectral response of the buried structures.

The diploma thesis deals with the determination of thickness and refractive index of thin organic films using image analysis. In the theoretical part there are described principles of the methods, which are used to prepare the films (spin coating, inkjet printing, vapour deposition), the characteristics of thin films, ways of finding out the thickness and refractive index of substances (weight methods, electric methods, method based on measurement of absorption coefficient of light, interference microscopy, ellipsometry) and also image analysis (harmonic and wavelet analysis). Interference microscope Epival - Interpako (Carl Zeiss Jena), digital camera Nikon Coolpix 5400 and computer were used for the determination of thickness and refractive index. The thicknesses of layers were set on the basis of interference images of edges and grooves – both from the side of the metal contact and the side of underlying glass. The refractive indices of thin layers were then set using the recorded figures. In the final part of the thesis there are discussed the results of interference images photographed along the full length of the aluminium contact which are used for measuring electrical characteristics of DPP structures. The produces are thicknesses and refractive indices of individual layers.

The dissertation presents theoretical and experimental studies on the physical origin of the signal in photothermal microscopy of single particles. This noninvasive optical far field microscopy scheme allows the imaging and detection of single absorbing nanoparticles. Based on a heat-induced pertur- bation in the refractive index in the embedding medium of the nanoscopic absorber, a corresponding probe beam modification is measured and quantified. The method is well established and has been applied since its first demonstration in 2002 to the imaging and characterization of various absorbing particle species, such as quantum dots, single molecules and nanoparticles of different shapes. The extensive theoretical developments presented in this thesis provide the first quantitative assess- ment of the signal and at the same time enlarge its phenomenology and thereby its potential. On the basis of several approximation schemes to the Maxwell equations, which fundamentally gov- ern the interaction of light with inhomogeneities, several complementing models are devised which describe the photothermal signal both qualitatively and quantitatively. In succession an interdepen- dent and self-consistent set of theoretical descriptions is given and allows important experimental consequences to be drawn. In consequence, the photothermal signal is shown to correspond to the action of a nanoscopic (thermal) lens, represented by the spherically symmetric refractive index pro- file n(r) which accompanies the thermal expansion of the absorber’s environment. The achieved quantification allows the direct measurement of absorption cross-sections of nanoparticles. Further, a qualitatively new phenomenology of the signal is unraveled and experimentally demonstrated. The separate roles of the probing and the heating beams in photothermal microscopy is dismantled and the influence of their relative alignment shown to allow for a controlled adjustment of the effective detection volume. For the first time, both positive and negative signals are demonstrated to occur and to be the characteristic signature of the lens-like action on the probe beam. The detection of the probe beam’s modification is also shown to sensitively depend on the aperture used in the detection chan- nel, and a signal optimization is shown to be feasible. Also, a generalization of the detectable signal via the use of a quadrant photodiode is achieved. Specifically, measuring the far field beam deflec- tion the result of the beam passing the lens off-center manifests in a laterally split detection volume. Hereby, finally each classical photothermal spectroscopic techniques has been shown to possess its microscopic counterpart. Central to the understanding of this generalized and new phenomenology is a scalar wave-optical model which draws an analogy between the scattering of a massive particle wave-packet by a Coulomb potential and the deflection of a focused beam by a photonic potential connected with the thermal lens. The significance of the findings is demonstrated by its methodological implications on photother- mal correlation spectroscopy in which the diffusion dynamics of absorbing colloidal particles can be studied. The unique split focal detection volumes are shown to allow the sensitive measurement of a deterministic velocity field. Finally, the method is supplemented by a newly introduced sta- tistical analysis method which is capable of characterizing samples containing a heterogeneous size distribution.:Contents Bibliographic description Abbreviations 1 Introduction 2 Theoretical Background 2.1 The current literature on the subject of the photothermal signal 2.2 Thermal conduction, and the temperature field around heated nanoparticles 2.3 The linear thermo-refractive response and the thermal lens 2.4 MAXWELL equations and approximation schemes 2.4.1 The MAXWELL equations 2.4.2 HELMHOLTZ equations 2.4.3 Paraxial HELMHOLTZ equation for the field components 2.4.4 Geometrical optics and the eikonal ansatz 2.5 Diffraction and the optical resolution limit in far field microscopy 2.5.1 Transmission scanning microscopy 2.5.2 Point spread functions and aberrations 2.5.3 Scalar diffraction approximation for weakly focused beams 2.5.4 Vectorial diffraction for highly focused electromagnetic fields 2.5.5 Theoretical description of transmission signals 2.6 Elastic scattering of light 2.6.1 Overview of optical elastic scattering theory 2.6.2 The integral equation of potential scattering and the BORN approximation 2.6.3 The generalized LORENZ-MIE theory 2.6.4 The electromagnetic fields 2.6.5 Description of the incident field: beam shape coefficients 2.6.6 Multilayered scatterers 2.6.7 POYNTING vector and field decomposition 2.6.8 Energy balance & total cross-sections 2.6.9 Optical theorem & the extinction paradox 2.6.10 Small particle scattering: the RAYLEIGH-limit 2.7 Optical properties of gold nanoparticles & Surface plasmon resonances 2.7.1 Dielectric function of gold 2.7.2 Total cross-sections of plasmonic nanoparticles properties of gold nanoparticles & Surface plasmon resonances 2.8 (Hot) BROWNian motion, diffusion and their statistical analysis 2.8.1 (Hot) BROWNian motion 2.8.2 Diffusion and correlation analysis 2.8.3 Methods regarding the signal statistics of diffusing tracer particles 2.9 RUTHERFORD scattering of charged particles 2.9.1 Classical RUTHERFORD scattering 2.9.2 Quantum mechanical COULOMB scattering 3 Experimental Setup 3.1 Sample preparation 3.2 Photothermal microscopy setup 4 Photothermal Imaging: Results and Discussion 4.1 MAXWELL equations: Exact treatment of the PT signal 4.1.1 Angularly resolved powers: Fractional cross-sections 4.1.2 Incident power and background normalization 4.1.3 Fractional scattering and extinction cross-sections (off-axis) 4.1.4 Fractional scattering and extinction cross-sections (on-axis) 4.1.5 Small particle approximation(on-axis) 4.1.6 General properties of transmission scans 4.1.7 The thermal lens n(r) in the MIE-scattering framework 4.1.8 The photothermal signal F in the MIE scattering framework 4.2 Geometrical optics: Photonic RUTHERFORD scattering (ray optics) 4.2.1 FERMAT’s principle for a thermal lens medium 4.2.2 Gaussian beam transformation by a thermal lens 4.2.3 Experiments using weakly focused, i.e. nearly Gaussian beams 4.3 HELMHOLTZ equation: Photonic RUTHERFORD scattering (wave optics) 4.3.1 Plane-wave scattering 4.3.2 Focused beam scattering 4.3.3 Connection to the far field 4.3.4 Photothermal Rutherford scattering microscopy 4.3.5 Photothermal half-aperture measurements 4.4 Paraxial HELMHOLTZ equation: FRESNEL diffraction by a thermal lens 4.4.1 The diffraction integral and the phase mask for a thermal lens 4.4.2 The photothermal signal expressed via the image plane field 4.4.3 Experimental demonstration of the signal inversion 4.4.4 Connection to photothermal RUTHERFORD scattering 4.5 Plane-wave extinction & scattering by a thermal lens 4.5.1 The BORN approximation for the ideal and time-dependent thermal lens 4.5.2 The eikonal approximation for the ideal thermal lens and x>>1 4.5.3 Lessons to be learned from plane-wave scattering by thermal lenses 4.6 What is a lens? And is n(r) a lens? 5 Methodological Applications of the Results 5.1 Generalized photothermal correlation spectroscopy (incl. twin-PhoCS) 5.2 Photothermal signal distribution analysis (PhoSDA) 6 Summary and Outlook 6.1 Summary of the results 6.2 Outlook 7 Appendix 7.1 Material parameters 7.2 Calculation parameters 7.3 Interactive simulation scripts (Processing) 7.4 Vectorial scattering in the BORN-approximation 7.5 Details regarding the scattering framework 7.5.1 Connection between Gmn,TE,TM of Ref.1 and gmn,TE,TM in the GLMT 7.5.2 Off-axis BSCs including aberration (single interface) 7.5.3 Details on the incidence power Pinc 7.5.4 Details on the incidence power Pinc for arbitrary beams 7.5.5 Explicit expressions for the spherical field components of Es,i and Hs,i 7.5.6 Note on the time-dependence and the corresponding sign-conventions in M 7.5.7 Recurrence relation for Pn and tn 7.5.8 Gaussian beam shape coefficients: Off-axis 7.5.9 Multilayered Scatterer 7.5.10 POYNTING-vector and energy flow fields 7.5.11 Convergence 7.5.12 Further evaluations in the GLMT framework 7.5.13 Diffraction model: Comparison of angular PT signal pattern to the GLMT 7.6 Details on geometrical optics models 7.6.1 Geometrical optics: Exact solution r(f) for |bx|<1 7.6.2 Correspondences in photonic and partile RUTHERFORD scattering 7.6.3 On the difference in the definition of optical energy 7.6.4 Ray-opticsphotothermalsignal 7.6.5 Thick lens raytracing and the equivalent lens shape for a given aberration 7.7 Thermal lens around a wire of radius R 7.8 Twin-PhoCS: Graphic illustration of the CCF integrand Curriculum Vitae Publications Declaration Acknowledgements List of Tables List of Figures Bibliography

Ce manuscrit de thèse présente l’étude des techniques de super-résolution latérale en nanoscopie optique, qui est une des nouvelles techniques d'imagerie haute résolution, aujourd'hui largement utilisée en biophysique et en imagerie médicale, pour imager et caractériser des nanostructures, tout en conservant les avantages de l'imagerie optique en champ lointain comme un vaste champ, la visualisation et l’analyse en temps réel…Un des défis futurs de la microscopie 3D super-résolue est d’éviter l’utilisation des marqueurs fluorescents. La microscopie interférométrique fait partie des techniques d’imagerie 3D sans marquage permettant la détection de nanostructures. Pour améliorer le pouvoir de détection de ce système optique, un premier protocole de traitement d’images a été développé et implémenté, permettant ainsi de révéler des structures initialement non mesurables. Puis, pour améliorer la résolution latérale du système, une nouvelle technique combinant l’interférométrie et le principe du nano-jet photonique a été développée permettant l’observation d’objets de taille inférieure à la limite de diffraction de l’instrument optique. / This manuscript presents the study of the lateral super-resolution techniques in optical nanoscopy, which is a new high-resolution imaging method now widely used in biophysics and medical imaging, to observe and measure nanostructures, with the advantages of far field optical imaging, such as a large field of view, visualization and analysis in real time…One of the future challenges of 3D super resolution microscopy is to avoid the use of fluorescent markers. Interferometric microscopy is a 3D label-free imaging technique enabling the detection of nanostructures. To improve the detection capability of this optical system, a first version of a protocol composed of image processing methods was developed and implemented, revealing structures initially unmeasurable. Then, to improve the lateral resolution of the system, a new technique combining interferometry and the principle of the photonic nano-jet has been developed, thus allowing the observation of objects of a size smaller than the diffraction limit of the optical instrument.