Michał Dudek

Tomographic phase microscopy of living three-dimensional cell cultures

Abstract. A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a “3-D cluster” as well as subcellular organelles like the nucleoli and local internal refractive index changes.

Noise suppressed optical diffraction tomography with autofocus correction

We propose a novel tomographic measurement approach that enables a noise suppressed characterization of microstructures. The idea of this work is based on a finding that coherent noise in the input phase data generates an artificial circular structure whose magnitude is the highest at the centre of tomographic reconstruction. This method decreases the noise level by applying an unconventional tomographic measurement configuration with an object deliberately shifted with respect to the rotation axis. This enables a spatial separation between the reconstructed sample structure and the area of the largest refractive index perturbations. The input phase data defocusing that is a by-product of the introduced modification is numerically corrected with an automatic focus correction algorithm. The proposed method is validated with simulations and experimental measurements of an optical microtip.

Problems and Solutions in 3-D Analysis of Phase Biological Objects by Optical Diffraction Tomography

Optical Diffraction Tomography is a technique for retrieving a 3-dimensional refractive index distribution from phase objects without destroying the structure of the samples. In the article we discuss the selection and implementation of full and limited angle version of tomographic reconstruction processes together with the analysis of different methods for gathering projections. We present two efficient implementations of full and limited angle tomographic systems including total processing paths and providing the examplary results of 3-D refractive index determination measurements of biological samples.

Interferometric and tomographic investigations of polymer microtips fabricated at the extremity of optical fibers

In this paper we present a simple method of manufacturing micrometer-sized polymer elements at the extremity of both single mode and multimode optical fibers and its possible modifications in order to provide requested functionalities. We show that the knowledge about 3D distribution of refractive index and birefringence in these elements is required and that interferometric and elastooptics tomography are the methods which provide these data. Exemplary polymer microtips manufactured from the polymeric material with different concentration of heptafluorobutyric acid are investigated in tomographic systems and the obtained results are discussed in reference to the theoretically expected refractive index distributions.

Tomographic and numerical studies of polymer bridges between two optical fibers for telecommunication applications

Abstract. We present the tomographic study of the refractive index distribution in polymer bridges between two optical fibers. Detailed refractive index maps are needed in order to improve the technological process for manufacturing those bridges and to achieve a lower return loss. At first, the technological process of the fabrication of bridges through photopolymerization is presented. The interferometric measurements of reference fibers used to produce those bridges and two series of microbridges are performed experimentally in the visible (VIS; 632.8 nm) and infrared (IR; 1550 nm) wavelength regions. The relation between the VIS and IR results is determined, which allows performing tomographic measurements in more accurate conditions in the VIS spectrum. The experimentally obtained refractive index distributions in the microbridges are used for modeling the insertion and return losses, which are compared with the real loss obtained for the produced microbridges. This knowledge will be used for better understanding the manufacturing process and its further optimization.

Holographic method for capillary induced aberration compensation for 3D tomographic measurements of living cells

In this paper we present a method for numerical correction of phase images captured in a digital holographic microscopy (DHM) setup adapted to tomographic measurement of biological objects. The purpose of the correction is a removal of the object wave deformation associated with a fluid filled fiber capillary, which is used in DHM system to enable manipulation of a specimen. The proposed correction procedure is based on a simple concept of the phase subtraction, preceded by an estimation of the aberration profile using areas of a hologram that have not been affected by the object. The phase subtraction methodology, developed on the ground of the thin element approximation, is very effective in the visual enhancement of phase images; however, its application to quantitative measurement of micro-objects is questionable. Therefore, in this paper we verify the possible use of the phase subtraction methodology in DHM by performing a numerical experiment, supported with the finite difference time domain method (FDTD), which allows us to identify the residual error of the correction. The FDTD computation reveals that the phase subtraction methodology is insufficient to properly remove the influence of a capillary, in particular to compensate for two effects associated with the focusing properties of the aberration: a transversal shift of the image and the change of its magnification. Nevertheless, the possibility of the visual improvement of holographic images of a living human leukemia cell using the outlined method is demonstrated.

Polymer Microtips at Different Types of Optical Fibers as Functional Elements for Sensing Applications

In this paper, we report on a simple process of fabrication of micrometer-sized polymer elements (microtips) at the extremities of different type optical fibers, including: standard single mode (SMF-28E+, SM-450), photonic crystal (LMA-10), and polymer (GIPOF-62) ones. The method of microtips manufacturing is based on the phenomenon of photopolymerization which is successfully used for the first time, to the best of our knowledge, to manufacture microtips at photonic crystal and polymer fibers. We discuss the influence of the initial process parameters on the final microtip characteristics (i.e., length, diameter, and profile). The potential applications of such polymer microtips are near-field scanning optical microscopy, coupling light sources with fibers, and sensing.

Microtips at photonic crystal fibers as functional elements for near-field scanning optical microscopy probes

We present the process of the microtip fabrication at the LMA-10 fiber designed for near-field scanning optical microscopy probes. Facilitation of manufacturing procedure as well as proper focusing conditions of such elements are the main advantages of such microelements production.

Optical properties of polymer microtips investigated with workshop tomographic system

We present a novel methodology for optical fiber polymer microtip manufacturing ant testing, which supports the structure optimization process through utilization of an optical diffraction tomography system based on the lateral shear digital holographic microscope. The most important functional parameter of an optical fiber microtip is the output beam distribution in the far-field region, which depends on geometrical properties and refractive index distribution within the microtip. These factors, in turn, are determined by the optical power distribution of the actinic light and the exposition time during the photopolymerization process. In order to obtain a desired light field distribution we propose to govern the manufacturing process by a hybrid opto-numerical methodology, which constitutes a convenient feedback loop for modification of the fabrication parameters. A single cycle of the proposed scheme includes numerical modeling, tomographic measurements and modifications of fabrication process. We introduced the real values of three-dimensional refractive index distribution of microtips into the finite-difference time-domain (FDTD) simulations, which leaded to controlled modification of technology parameters and finally to improvement of a functional parameter of microtips.

Problems and Solutions in Tomographic Analysis of Phase Biological Objects

Digital holographic microscopy (DHM), when applied to semitransparent samples such as living cells, provides accurate measurements of phase shift resulting from a mean refractive index accumulated over the cellular thickness [1-5]. However a single shot holography is inadequate to provide true 3D reconstruction of a cell structure although such reconstruction is required to solve several hot biomedical topics including: label-free analysis of living cells and tissues, characterization of physical processes in cellular biophysics, extended studies in vascular and tumor biology, as well as recognition and monitoring of bacteria colonies. It has been proved by many groups that the solution can be provided by combining digital holography with tomographic techniques. The resultant method, often referred as optical diffraction tomography (ODT) [6-9] requires multiple complex object field captured for different illumination directions with respect to the sample and latter tomographic reconstructing of a three-dimensional distribution of refractive index. The projections are obtained through varying the illumination direction or rotating the specimen. Number of various approaches has been so far reported to deal with this problem including trapping a specimen with micropipette [10] or optical tweezers [11] or altering the angle of illumination using a galvanometer scanning mirror [12] and multiple fibre optics illumination [13]. In this study we present a cost-efficient, sample-rotational-based tomography module for 3D label-free quantitative live cell measurements based on a hollow optical fiber as the sample cuvette. Several problems connected with implementation of the full tomographic reconstruction process are discussed including tracking of a cell position, minimizing errors associated with a fluid field fiber capillary of a sample cuvette, determination of absolute values of refractive index and the possibility of reconstruction from a limited angle of projections. We also show the examples of exciting results which can be obtained with this tool.