Sabine Przibilla

Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes

Abstract. The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

Application of 3D tracking, LED illumination and multi-wavelength techniques for quantitative cell analysis in digital holographic microscopy

Digital Holographic Microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Partially coherent light sources and multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the utilization of light emitting diodes (LEDs) as well as the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that LEDs and multi-wavelength techniques enhance the axial resolution in inspection of reflective surfaces and quantitative digital holographic cell imaging.

Self interference digital holographic microscopy approach for inspection of technical and biological phase specimens

Quantitative holographic phase contrast imaging enables high-resolution inspection of reflective surfaces and technical phase specimen as well as the minimally invasive analysis of living cells. However, a drawback of many experimental arrangements is the requirement for a separate reference wave which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, a self interference digital holographic microscopy (DHM) approach was explored which only requires a single object illumination wave. Due to the Michelson interferometer design of the proposed setup two wave fronts with an almost identical curvature are superimposed. This results in a nearly ideal pattern of spatial off-axis carrier fringes and a constant interferogram contrast in the hologram plane. Moreover, the hologram evaluation with spatial phase shifting reconstruction algorithms and Fourier transformation-based spatial filtering methods as well as the integration of DHM in common research microscopes is simplified. Furthermore, the use of laser light sources with a short coherence length is enabled. The applicability of the proposed self interference principle is illustrated by data from the analysis of technical and biological phase specimens. The obtained results demonstrate that the method prospects to be a versatile tool for quantitative phase contrast imaging.

Tonicity induced changes in volume and refractive index of suspended cells quantified with digital holographic microscopy

The paper presents the way that colour can serve solving the problem of calibration points indexing in a camera geometrical calibration process. We propose a technique in which indexes of calibration points in a black-and-white chessboard are represented as sets of colour regions in the neighbourhood of calibration points. We provide some general rules for designing a colour calibration chessboard and provide a method of calibration image analysis. We show that this approach leads to obtaining better results than in the case of widely used methods employing information about already indexed points to compute indexes. We also report constraints concerning the technique. Nowadays we are witnessing an increasing need for camera geometrical calibration systems. They are vital for such applications as 3D modelling, 3D reconstruction, assembly control systems, etc. Wherever possible, calibration objects placed in the scene are used in a camera geometrical calibration process. This approach significantly increases accuracy of calibration results and makes the calibration data extraction process easier and universal. There are many geometrical camera calibration techniques for a known calibration scene [1]. A great number of them use as an input calibration points which are localised and indexed in the scene. In this paper we propose the technique of calibration points indexing which uses a colour chessboard. The presented technique was developed by solving problems we encountered during experiments with our earlier methods of camera calibration scene analysis [2]-[3]. In particular, the proposed technique increases the number of indexed points points in case of local lack of calibration points detection. At the beginning of the paper we present a way of designing a chessboard pattern. Then we describe a calibration point indexing method, and finally we show experimental results. A black-and-white chessboard is widely used in order to obtain sub-pixel accuracy of calibration points localisation [1]. Calibration points are defined as corners of chessboard squares. Assuming the availability of rough localisation of these points, the points can be indexed. Noting that differences in distances between neighbouring points in calibration scene images differ slightly, one of the local searching methods can be employed (e.g. [2]). Methods of this type search for a calibration point to be indexed, using a window of a certain size. The position of the window is determined by a vector representing the distance between two previously indexed points in the same row or column. However, experiments show that this approach has its disadvantages, as described below. * E-mail: A.Nowakowski@ire.pw.edu.pl Firstly, there is a danger of omitting some points during indexing in case of local lack of calibration points detection in a neighbourhood (e.g. caused by the presence of non-homogeneous light in the calibration scene). A particularly unfavourable situation is when the local lack of detection effects in the appearance of separated regions of detected calibration points. It is worth saying that such situations are likely to happen for calibration points situated near image borders. Such points are very important for the analysis of optical nonlinearities, and a lack of them can significantly influence the accuracy of distortion modelling. Secondly, such methods may give wrong results in the case of optical distortion with strong nonlinearities when getting information about the neighbouring index is not an easy task. Beside this, the methods are very sensitive to a single false localisation of a calibration point. Such a single false localisation can even result in false indexing of a big set of calibration points. To avoid the above-mentioned problems, we propose using a black-and-white chessboard which contains the coded index of a calibration point in the form of colour squares situated in the nearest neighbourhood of each point. The index of a certain calibration point is determined by colours of four nearest neighbouring squares (Fig.1). An order of squares in such foursome is important. Because the size of a colour square is determined only by the possibility of correct colour detection, the size of a colour square can be smaller than the size of a black or white square. The larger size of a black or white square is determined by the requirements of the exact localisation step which follows the indexing of calibration points [3]. In this step, edge information is extracted from a blackand-white chessboard. This edge information needs larger Artur Nowakowski, Wladyslaw Skarbek Institute of Radioelectronics, Warsaw University of Technology, Nowowiejska 15/19, 00-665 Warszawa, A.Nowakowski@ire.pw.edu.pl Received February 10, 2009; accepted March 27, 2009; published March 31, 2009 http://www.photonics.pl/PLP

Quantitative phase imaging-based refractive index determination of living cells using incorporated microspheres as reference

In quantitative phase-based live cell imaging the intracellular refractive index represents an important parameter. On one hand decoupling of the refractive index and the cell thickness is required, e. g., for reliable investigations on the cell morphology. On the other hand the cell refractive index and its spatial distribution is related to the concentration of the intracellular content. We explored a method to determine the mean refractive index of the cytoplasm with digital holographic microscopy (DHM). Microspheres that have been incorporated by living cells are used as reference in quantitative DHM phase contrast images from which the refractive index is obtained by a two dimensional fitting procedure. As many cells show a phagocytic behavior the method may be used with a variety of different cell types. Furthermore, as no modification of the experimental setup is required, the principle prospects to be used with several existing quantitative phase contrast imaging techniques.

Multi-wavelength digital holographic microscopy for high-resolution inspection of surfaces and imaging of phase specimen

Main drawbacks of using laser light in digital holographic microscopy (DHM) are coherent noise and parasitic reflections in the experimental setup as these disturbances affect the reconstructed images and restrict the measurement accuracy. Partially coherent light reduces such effects. On the other hand, the application of light sources with a low coherence length requires a precise alignment of the experimental equipment. Thus, it was investigated, if coherence properties of spectral broadened light sources can be generated synthetically with laser light. Therefore, amplitude and phase distributions are superposed that result from the reconstruction of digital holograms which are recorded separately at slightly different wavelengths. In this way, the robust alignment of a laser-based experimental setup due to long coherence lengths is combined with the noise reduction advantage of partial coherent light. By using a single fiber coupled tuneable laser the multi-wavelength approach can be used with already existing DHM setups, e. g., in combination with commercial microscopes. The performance of the method for the observation of phase objects is illustrated by results obtained from the topography analysis of reflective surfaces and from the application for quantitative phase contrast imaging of thin living tumor cells.

Manipulating intracellular refractive index for contrast-enhanced digital holographic imaging of subcellular structures

The online analysis of rapid cellular processes by morphological alterations strongly depends on the ability to rapidly visualize and to quantify cell shape and intracellular structures. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogenous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyzed the impact of specific intracellular refractive index manipulation by microinjection of refractive index changing agents on the DHM phase contrast. Glycerol was chosen as osmolyte, which combines high solubility in aqueous solutions and cellular compatibility. We present data showing that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle was proven by experiments inducing cell shrinkage and protein concentration. The integrity of cell membranes is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for DHM opens also prospects for application with other quantitative phase contrast imaging technologies.

3D Tracking and Multi-Wavelength Techniques for Digital Holographic Microscopy based Cell Analysis

Digital Holographic Microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that multi-wavelength techniques enhance the phase resolution in quantitative digital holographic cell imaging.

Simplified setup for imaging with digital holographic microscopy and enhanced quantitative phase contrast by osmotic stimulation of living cells

Many interferometry-based quantitative phase contrast imaging techniques require the generation of a coherent reference wave, which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, investigations on a simplified digital holographic microscopy approach that avoids a separate reference wave were performed. Results from live cell investigations demonstrate the capability of the method for quantitative phase contrast imaging. In further experiments the modification of the intracellular refractive index distribution by osmotic stimulation was analyzed. Data from human pancreas tumor cells show that by choice of suitable buffer solutions live cell imaging with enhanced quantitative phase contrast is achieved.