Analyzing the structure of a single cell based on its refractive index (RI) distribution is a common and valued approach, because it does not require any artificial markers. The RI is an inherent structural marker that can be quantified in three dimensions with optical diffraction tomography (ODT), an inverse scattering technique. This work reviews the theory of ODT and its implementation with an emphasis on single-cell analysis, identifying the Rytov approximation as the most efficient descriptor for light propagation. The accuracy of the reconstruction method is verified with in silico data and imaging artifacts associated with the inverse scattering approach are addressed. Furthermore, an experimental ODT setup is presented that consists of a bright-field microscope, a phase-imaging camera, and an optical trap combined with a microfluidic chip. A novel image analysis pipeline is proposed that addresses image corrections and frame alignment of the recorded data prior to the RI reconstruction. In addition, for a rotational axis that is tilted with respect to the image plane, an improved reconstruction algorithm is introduced and applied to single, suspended cells in vitro, achieving sub-cellular resolution.

Analyzing the structure of a single cell based on its refractive index (RI) distribution is a common and valued approach, because it does not require any artificial markers. The RI is an inherent structural marker that can be quantified in three dimensions with optical diffraction tomography (ODT), an inverse scattering technique. This work reviews the theory of ODT and its implementation with an emphasis on single-cell analysis, identifying the Rytov approximation as the most efficient descriptor for light propagation. The accuracy of the reconstruction method is verified with in silico data and imaging artifacts associated with the inverse scattering approach are addressed. Furthermore, an experimental ODT setup is presented that consists of a bright-field microscope, a phase-imaging camera, and an optical trap combined with a microfluidic chip. A novel image analysis pipeline is proposed that addresses image corrections and frame alignment of the recorded data prior to the RI reconstruction. In addition, for a rotational axis that is tilted with respect to the image plane, an improved reconstruction algorithm is introduced and applied to single, suspended cells in vitro, achieving sub-cellular resolution.