Transport of Intensity Equation based Quantitative Phase Imaging of Red Blood Cells

Abstract

This thesis reports the characterization of Red Blood Cell (RBC) morphology in a non-contact and label-free manner using a flexible, low-cost continuous imaging system. Detection of abnormalities in red blood cell properties, including shape, size, and number, can reveal a range of pathologies. A usual laboratory hematologic diagnosis consists of a complete blood count (CBC) and a peripheral blood smear (PBS) review. An Automated Hematology Analyzer reports the complete blood count, which includes Hematocrit, Hemoglobin content in RBCs and Total Count, Mean Cell Volume, Distribution width for each blood cell (RBC, WBC, Platelets). But they fail to provide any information about the cell shape, which is an essential property in determining cell morphology. A peripheral blood smear analysis involves imaging RBCs under a microscope and determining cell shape, although cell volume cannot be found as cell thickness remains unknown. Since cells are imaged in dry form and distributed non-uniformly, finding cell count using a PBS review is not possible. So, there is a need for a different technique that can be used to study red blood cell morphology effectively. Today one of the rapidly growing research fields in studying cell morphology and cell dynamics is Quantitative Phase Imaging (QPI). It combines advancements in optics, imaging theory, and computational methods to image phase information of the sample quantitatively. In this work, determination of RBC total count, MCV, and RDWusing a single, fast, portable, and cost-effective optical setup has been proposed. It involves quantifying the phase delay introduced by the red blood cells using a QPI method called the Transport of Intensity Equation (TIE). The application of TIE as a QPI method does not require complex setups or expensive components, unlike other QPI techniques. A partially coherent light beam from a conventional LED is used to illuminate a diluted blood sample loaded in a microfluidic channel. Through-focus intensity images of RBCs arranged in a monolayer are acquired using a low-cost continuous imaging system. Intensity images are processed using a Fast Fourier Transform (FFT) based Poisson solver to find a solution to the transport of intensity equation. The solution to TIE is the phase distribution of light at the focus. From phase, the thickness profile of each cell can be calculated, hence the cell volume. RBC total count is calculated by counting the cells in the given field of view. Cell shape is determined from the focal image. So the proposed system provides complete information about the red blood cells morphology at a much lower cost than hematology analyzers and peripheral blood smear analysis. The overall aim of this research project is to explore the potential of our imaging system combined with the TIE algorithm as a reliable tool for characterizing red blood cells morphology