Optical projection tomography as a tool to analyze 3D cell cultures of cardiomyocytes

This Master’s thesis addresses the issue of the possibility of the imaging and analysis of the live cardiomyocytes’ clusters using Optical projection tomography. Cardiomyocytes are heart muscle cells that play an important role in the beating of the heart. Research scientists have been imaging beating cardiomyocytes using traditional phase contrast microscopy techniques. The Optical projection tomography technique involves placing a biological sample in the refractive index matching bath, for instance distilled water for gel-based samples because of the matching refractive index value, after which the sample is illuminated with the bright field light and the images are acquired using either a LabView or an HCImageLive program. Since, the Optical projection tomography is relatively a new technology and it is deemed more optimized for larger and static samples such as tissues and embryos etc. therefore, it is quite challenging to prove the efficacy of the Optical projection tomography method for imaging smaller and faster aggregates of cardiomyocytes. At first, cardiomyocytes are differentiated and produced from the human induced pluripotent stem cells at Heart Group, BioMediTech, UTA. After differentiation, live cardiomyocytes are placed in 3D hydrogel and imaged using Optical projection tomography. The samples are imaged using customized LabView program and HCImageLive program. The imaging method deploys an acquisition of 400-1000 images of the samples on different focal planes. In this regard, samples are rotated at different focal angles to visualize the beating pattern at different angular positions. After the continuous acquisitions are obtained, the videos of the live cardiomyocytes are constructed from the continuous frames of images using ImageJ. Once, the videos of the beating cardiomyocyes are constructed, both qualitative and quantitative analyses are done on the beating aggregates using analysis tools on MATLAB and ImageJ plugins, PIV (Particle Image Velocimetry) and FTTC (Traction force microscopy). The analysis focuses on the traction forces of the live cardiomyocytes. Results show that the Optical projection tomography technique can be successfully used for the imaging and characterization of the beating clusters of cardiomyocytes. Furthermore, the images and videos obtained from the Optical projection tomography technique are good for analysis using different software tools