Daniel Carl

Parameter-optimized digital holographic microscope for high-resolution living-cell analysis

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.

Investigation of living pancreas tumor cells by digital holographic microscopy.

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 cells cytoskeleton. The results demonstrate that digital holographic microscopy is a quantitative phase contrast technique for living cells under conventional laboratory conditions.

Holographic interferometric microscopy systems for the application on biological samples

Holographic interferometric metrology allows a fast, non destructive and quantitative high resolution full field detection of optical path length changes. Furthermore, by utilization of modern CCD sensor technology and digital image process-ing algorithms an on-line application of these methods even on biological specimens is possible. In combination with a microscopic resolution this offers new possibilities for the detection of variations of shape, micro movements or refractive index changes e. g. for the marker free analysis of cellular samples. Three holographic interferometric systems for microscopy applications based on digital holography, (speckle) interferometry and photorefractive crystals as holographic recording medium are introduced. Results of investigations on test charts and biological samples to characterize and optimize the lateral resolution as well as the resolution of the detected phase difference changes are presented and discussed. Finally, the applicability of the developed measurement techniques on living cells is demonstrated.

Structural properties of liposomes from digital holographic microscopy

We have constructed liposomes from L alpha Phosphatidylcholine (PC) lipids, which are biomimetic lipids similar to those present in the membranes of mammalian cells. We propose an advance in the use of liposomes, such as for drug delivery, to incorporate into the liposomal membranes transport proteins that have been extracted from the lipid membranes of mammalian cells. In this paper, we describe the usage of a novel optical microscope to characterize the nanomechanical properties of these liposomes. We have applied the technique of digital holographic microscopy, using an instrument recently developed at the University of Münster, Germany. This system enabled us to measure quantitatively the structural changes in liposomes. We have investigated the deformations of these biomimetic lipids comprising these liposomes by applying osmotic stresses, in order to gain insight into the membrane environment prior to incorporation of cloned membrane transport proteins. This control of the nanomechanical properties is important in the stresses transmitted to mechanosensitive ion channels that we have incorporated into the liposomal membranes. These liposomes provide transporting vesicles that respond to mechanical stresses, such as those that occur during implantation.

Algorithm for fringe independent quantification of noise in wrapped phase distributions obtained by digital holography and speckle interferometry

The noise of phase distributions is an important parameter for the optimization of holographic and speckle interferometric setups as well as to quantify the measurement accuracy for the determination of optical path length changes. An algorithm for noise quantification of wrapped phase distributions is presented that is based on an enhanced sine-cosine filter with subsequent triangle function transformation. Compared to methods that calculate the standard deviation of raw phase data modulo 2π to smoothed data obtained by spatial filtering with a sine-cosine filter, an enhanced accuracy for noise quantification is achieved that is independent of fringe number and fringe orientation.