Charakterisierung von Methoden und Anwendungen der digitalholographischen Mikroskopie

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An off-axis setup for digital holographic microscopy in incident and transmission light arrangement for simultaneous high resolution full field amplitude and quantitative phase contrast microscopy is presented. Different kinds of algorithms for numerical reconstruction of the complex object wave are compared concerning their applicability to the microscopy arrangement. By combining a non-diffractive spatial phase shifting algorithm that performs reconstruction without the disturbing terms twin image and zero order with the numerical evaluation of the Fresnel- Kirchhoff diffraction integral by a convolution method we achieve best results. The simultaneous reconstruction of the object wave’s amplitude and phase from a single hologram requires a mathematical model of the spatial phase distribution within the hologram plane. An efficient numerical algorithm has been developed for determining the model’s parameters automatically and tested concerning its accuracy. Furthermore, the relation between the axial position of the object and the distance of reconstruction which is required for the quantitative evaluation of the reconstructed images and the application of a pure numerical focus is derived. Technical objects were used to quantify the lateral resolution and the phase resolution of the system and to optimize several parameters of the setup. Biological probes such as living cells are analyzed in transmission light arrangement. As a result the knowledge of the refractive index of the medium and the cell is required to derive the cell’s thickness from the reconstructed phase. Thus a special experimental method for the approximation of the integral refractive index of single cells from the reconstructed phase has been developed. Finally results of cell differentiation by morphological varieties as well as results of stimulated dynamic morphological changes are presented and discussed.