Quantiative biological microsocopy by digital holography

In this dissertation, improved techniques in digital holography, that have produced high-resolution, high-fidelity images, are discussed. In particular, the angular spectrum method of calculating holographic optical field is noted to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. It is observed that spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. In the angular spectrum method, the reconstruction distance does not have a lower limit, and the off-axis angle between the object and reference waves can be lower than that of the Fresnel requirement, while still allowing the zero-order background to be cleanly separated. Holographic phase images are largely immune from the coherent noise commonly found in amplitude images. With the use of a miniature pulsed laser, the resulting images have 0.5um diffraction-limited lateral resolution and the phase profile is accurate to about several nanometers of optical path length. Samples such as ovarian cancer cells (SKOV-3) and mouse-embryo fibroblast cells have been imaged. These images display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. This technique clearly exceeds currently available methods in phase-contrast opticalmicroscopy in both resolution and detail and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available. Furthermore, we also demonstrate that phase imaging digital holographic movies provide a novel method of non-invasive quantitative viewing of living cells and other objects. This technique is shown to have significant advantages over conventional microscopy