Three-Dimensional Microscopy by Laser Scanning and Multi-Wavelength Digital Holography

This dissertation presents techniques of three-dimensional microscopy. First, an economical method of microscopic image formation that employs a raster-scanning laser beam focused on a sample, while non-imaging detector receives the scattered light is presented. The images produced by this method are analogous to the scanning electron microscopy with visible effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the system allows for a greater flexibility, as the variety of optical detectors, such as PMT and position-sensitive quadrant photodiode can be used to acquire images. The system demonstrates a simple, low-cost method of achieving the resolution on the order of a micron. A further gain in terms of resolution and the depth of focus by using Bessel rather than Gaussian beams is discussed. Then, a phase-imaging technique to quantitatively study the three-dimensional structure of reflective and transmissive microscopic samples is presented. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of porous coal samples and SKOV-3 ovarian cancer cells are presented