Problems and Solutions in Tomographic Analysis of Phase Biological Objects

Introduction

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.