Digital holographic microscopy provides new facilities for contactless and marker-free quantitative phase contrast imaging. In this work, a digital holographic microscopy method for the integral refractive index determination of living single cells in cell culture medium is presented. Further, the obtained refractive index information is applied to full field thickness and shape determination of adherent pancreas tumor cells, as well as for analysis of drug-induced dynamic changes of a single cell's cytoskeleton. The results demonstrate that digital holographic microscopy is a quantitative phase contrast technique for living cells under conventional laboratory conditions.
A parameter-optimized off-axis setup for digital holographic microscopy is presented for simultaneous, high-resolution, full-field quantitative amplitude and quantitative phase-contrast microscopy and the detection of changes in optical path length in transparent objects, such as undyed living cells. Numerical reconstruction with the described nondiffractive reconstruction method, which suppresses the zero order and the twin image, requires a mathematical model of the phase-difference distribution between the object wave and the reference wave in the hologram plane. Therefore an automated algorithm is explained that determines the parameters of the mathematical model by carrying out the discrete Fresnel transform. Furthermore the relationship between the axial position of the object and the reconstruction distance, which is required for optimization of the lateral resolution of the holographic images, is derived. The lateral and the axial resolutions of the system are discussed and quantified by application to technical objects and to living cells.
A novel implementation of lensless multiwavelength digital holography with autocalibration of temporal phase shifts and artificial wavelength is presented. The algorithm we used to calculate the phase shifts was previously proposed [Opt. Lett.29 183 (2004)] and, to our knowledge, is now used for the first time in lensless holography. Because precise knowledge of the generated artificial wavelength is crucial for absolute measurement accuracy, a simple and efficient method to determine the artificial wavelength directly is presented. The calibration method is based on a simple modification of the experimental setup and needs just one additional image acquisition per wavelength. The results of shape measurement of a metallic test object with a rough surface and steep edges are shown and the measurement accuracy is discussed.
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