Automated Methods for Analysis of Three-Dimensional Morphology and Dynamics in Living Cells With a Label-Free Digital Holographic Microscope

Abstract

The most common type of microscope is the optical two-dimensional (2D) microscope. This is an optical device containing one or more lenses producing an enlarged image of a sample placed in the focal plane of the first lens. Optical microscopes have refractive glass and occasionally of plastic or quartz, to focus light into the eye or another light detector. The usage of the light microscopes has been around and will be. However, there is a big flaw in using 2D microscopes. This microscopic system suffers from losses of quantitative detailed information about 3rd dimension (thickness) of transparent or semitransparent microscopic samples. The problem is not limited only to this one. There are cases in which samples are transparent or semi-transparent. In these cases the light cannot be reflected thus a high contrast image will not be produced. This can falsify cell studies and results to incorrect outputs. To overcome these problems, three-dimensional imaging techniques are suggested. Among these techniques, digital holographic microscopy (DHM) has been successfully applied in a range of application areas. Indeed, DHM is not destructive for specimens and sample can be investigated many times and by any other optical methods while the electron microscopy methods are destructive. Another advantage of using DHM is that we can investigate the cells in single-cell level. Moreover, quantitative phase image obtained by DHM enables one to measure characteristic properties of cells such as thickness, volume, surface area and projected surface area. The usage of DHM can be extended to time-lapse imaging (four dimensional imaging; last dimension is the time). Time lapse imaging enables us to study cell changes or growths in different time stamps and monitor it. In this dissertation, we would like to address different automated techniques and methods which can enable us to study live cells in non-destructive manner and single-cell level. Several approaches for studying and analyzing live cells are proposed and applied. To do so, two subject cells are concerned. The first cell type is red blood cell (RBC) and the second one is cardiomyocyte. Each type of these cells has different cell structure. In the case of RBC, DHM is perfectly suited for different experiments and it can provide thickness of the cell in nanometer accuracy. The reason is that cell has no nuclei and organelles. So, we are able to study different characteristic of the cell. Cardio cell is well suited for time-lapse studies. The reason is that optical path difference can be measured by DHM device. This can help us studying cell structure changes during different states of beating. Several experimental results are conducted and in some cases results are compared with the chemical experiments to show the feasibility of the proposed methods.