Phase control and measurement in digital microscopy

The ongoing merger of the digital and optical components of the modern microscope is creating opportunities for new measurement techniques, along with new challenges for optical modelling. This thesis investigates several such opportunities and challenges which are particularly relevant to biomedical imaging. Fourier optics is used throughout the thesis as the underlying conceptual model, with a particular emphasis on three–dimensional Fourier optics. A new challenge for optical modelling provided by digital microscopy is the relaxation of traditional symmetry constraints on optical design. An extension of optical transfer function theory to deal with arbitrary lens pupil functions is presented in this thesis. This is used to chart the 3D vectorial structure of the spatial frequency spectrum of the intensity in the focal region of a high aperture lens when illuminated by linearly polarised beam. Wavefront coding has been used successfully in paraxial imaging systems to extend the depth of field. This is achieved by controlling the pupil phase with a cubic phase mask, and thereby balancing optical behaviour with digital processing. In this thesis I present a high aperture vectorial model for focusing with a cubic phase mask, and compare it with results calculated using the paraxial approximation. The effect of a refractive index change is also explored. High aperture measurements of the point spread function are reported, along with experimental confirmation of high aperture extended depth of field imaging of a biological specimen. Differential interference contrast is a popular method for imaging phase changes in otherwise transparent biological specimens. In this thesis I report on a new isotropic algorithm for retrieving the phase from differential interference contrast images of the phase gradient, using phase shifting, two directions of shear, and non–iterative Fourier phase integration incorporating a modified spiral phase transform. This method does not assume that the specimen has a constant amplitude. A simulation is presented which demonstrates good agreement between the retrieved phase and the phase of the simulated object, with excellent immunity to imaging noise.