Cellular Dynamics and Three-Dimensional Refractive Index Distribution Studied with Quantitative Phase Imaging

We present in this PhD thesis work various applications of digital holographic microscopy (DHM), an imaging technique based on coherent illumination which enables the recovery of the full complex wavefront, i.e. the amplitude and phase of a wave field which interacted with a specimen. The possibility to retrieve the phase information with DHM allows to measure surfaces with nanometric accuracy, or to employ it as an endogenous quantitative signal to assess the morphology of biological specimens. The technique has been developed during the past fifteen years to reach nowadays a mature state, where it can be used routinely for metrology applications for example. We study in this work advanced applications by taking advantage of this technique, while focusing on a specific measurement method of DHM, namely the off-axis configuration, which makes it possible to measure the complex wave field with one-shot capability through spatial encoding, thus enabling real-time detection. In a first part, we develop mathematical methods based on the fundamental model of holographic recording to suppress the so-called zero-order, which consists in intensity terms that coherent detection must suppress for complex wave retrieval. In the particular case of off-axis holography, the zero-order terms usually limit the spatial resolution because of the spatial encoding of the coherent signal. We first develop an iterative method which uses the fundamental relations between coherent and incoherent detection, in order to gradually suppress the zero order terms. In a second stage, we develop a non-iterative filtering method, based on nonlinear operators. The technique is based on the transfer to another filtering space through the use of the logarithm, and enables intrinsic suppression of the zero-order terms. Both methods present the advantage of not relying on any approximation, and are thus general for any off-axis holographic configuration. We show their applicability on various hologram types, and demonstrate that in the context of microscopy, their use can increase the spatial resolution of holography, in order to reach diffraction-limited imaging for any magnification. In a second part, we study potential applications of three-dimensional imaging through coherent detection by employing multiple acquisitions with a new scanning method. The coupling of tomographic reconstruction and quantitative phase imaging showed great potential in various published works, yielding to quantitative 3D refractive index distribution measured within biological specimens, and super-resolution imaging through synthetic aperture formalism. These methods are however still subjects to many issues, in particular due to practical limitations such as mechanical imprecision in the measurement protocols and the availability of flexible reconstruction algorithms. We study a new data acquisition method which eliminates the necessity of any scanning of the illumination pattern or object rotation dur