Diseases of the myocardium place a large and growing economic burden on the United States' healthcare system. Advancements in pharmaceutical research and clinical and community practices have stemmed mortality rates from the category of diseases, but prevalence and rates of hospitalization remain on the rise. Regenerative and molecular cardiology offer different yet intersecting approaches to understanding and treating these diseases. In regenerative cardiology, one vision is to directly regenerate damaged myocardium via the transplantation of stem cell-derived cardiomyocytes. In molecular cardiology, understanding the signaling and regulatory pathways that allow diseases such as hypertension and heart failure to feed-forward are paramount. To address specific needs within both fields, we develop the nonlinear optical process of second harmonic generation (SHG) as an enabling imaging technology, with the aim of allowing new and unique experiments to be performed.
Within the scope of regenerative cardiology, a specific technical problem is that there is no widely accepted, effective method for purifying pluripotent stem cell-derived cardiomyocytes (PSC-CMs) for clinical applications. Similarly, there is no effective method for selecting PSC-CMs based on their maturity. We show that SHG can be used to identify and characterize PSC-CMs without the use of exogenous labels. SHG is detectable in PSC-CMs and not detectable in PSCs. Furthermore, SHG signal strength is dependent on PSC-CM maturity and is retained while PSC-CMs are in suspension. The results demonstrate that SHG has the potential to be harnessed for the maturity-based selection of PSC-CMs, but the implementation of nonlinear optical processes in high-speed cytometry is not trivial. In order efficiently deliver enough intensity to the cell sample to excite SHG, we develop a Bessel beam-based excitation scheme suitable for use with microfluidic cell sorters. It is demonstrated that a Bessel beam light sheet can excite detectable SHG in neonatal cardiomyocytes as they flow through a microfluidic channel. The results point toward the feasibility of constructing an SHG-activated microfluidic cell sorter for purifying and sorting PSC-CMs.
In molecular cardiology, a hypothesis has emerged from recent research is that there exist local non-uniformities in the structure and function of cardiomyocytes, and that these non-uniformities can drive forward disease processes in cardiomyocytes. Testing such a hypothesis requires a method for visualizing multiple aspects of cardiomyocyte biology with high resolution, in real time and with minimal perturbation, yet no appropriate methods exist. Here, we develop a multimodal technique that combines SHG with two photon fluorescence (TPF) of a fluorescent calcium indicator in order to visualize calcium-contraction coupling at the single sarcomere level. In live cardiomyocytes, we conduct multimodal SHG-TPF line scans along portions of myofilaments at a rate of 100-300 Hz, and with careful post-processing and analysis of the data, we show that it is indeed possible to correlate local calcium releases to the development of local strain in myofilaments. Future work will apply the technique in the study of animal models of hypertensive heart disease.
|Advisor:||Matthews, Dennis L., Chan, James W.|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 75/01(E), Dissertation Abstracts International|
|Subjects:||Biomedical engineering, Optics|
|Keywords:||Cardiomyocyte purification, Label free, Local strain, Second harmonic generation, Stem cells|
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