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Transient absorption imaging of hemeprotein in fresh muscle fibers




Wang, Erkang, author
Wilson, Jesse, advisor
Bartels, Randy, committee member
Krapf, Diego, committee member
Tobet, Stuart, committee member

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Mitochondrial diseases affect 1 in 4000 individuals in the U.S. among adults and children of all races and genders. Nevertheless, these diseases are hard to diagnose because they affect each person differently. Meanwhile the gold standard diagnosis methods are usually invasive and time- consuming. Therefore, a non-invasive and in-vivo diagnosis method is highly demanded in this area. Our goal is to develop a non-invasive diagnosis method based on the endogenous nonlinear optical effect of the live tissues. Mitochondrial disease is frequently the result of a defective electron transport chain (ETC). Our goal is to develop a non-invasive way to measure redox within the ETC, specifically, of cytochromes. Cytochromes are iron porphyrins that are essential to the ETC. Their redox states can indicate cellular oxygen consumption and mitochondrial ATP production. So being able to differentiate the redox states of cytochromes will offer us a method to characterize mitochondrial function. Meanwhile, Chergui's group found out that the two redox states of cytochrome c have different pump-probe spectroscopic responses, meaning that the transient absorption (TA) decay lifetime can be a potential molecular contrast for cytochrome redox state discrimination. Their research leads us to utilize the pump-probe spectroscopic idea to develop a time-resolved optical microscopic method to differentiate not only cytochromes from other chemical compounds but also reduced cytochromes from oxidized ones. This dissertation describes groundbreaking experiments where transient absorption is used to reveal excited-state lifetime differences between healthy controls and an animal model of mitochondrial disease, in addition to differences between reduced and oxidized ETC in isolated mitochondria and fresh preparations of muscle fibers. For our initial experiments, we built a pump-probe microscopic system with a fiber laser source, producing 530nm pump and 490nm probe using a 3.5kHz laser scanning rate. The pulse durations of pump and probe are both 800fs. For the preliminary results, we have successfully achieved TA decay contrast between reduced and oxidized cytochromes in solution form. Then we have achieved SNR enhanced pump-probe image of BGO crystal particles with the help of the software- based adaptive filter noise canceling method. We also have installed a FPGA-based adaptive filter to enhance the pump-probe signals of the electrophoresis gels that contain different mitochondrial respiratory chain supercomplexes. However, because the noise floor was still 30 dB higher than shot noise limit, cytochrome imaging in live tissues was still problematic. We then built another pump-probe microscope with a solid- state ultrafast laser source. In that way, we do not need to worry about laser relative intensity noise (RIN) anymore, since the noise floor of the solid-state laser source can reach the shot noise limit at MHz region. One other advantage of the new laser source is that it can provide one tunable laser output that can be directly converted to the probe pulse with tunable center wavelength. Its tunability can cover the entire visible spectrum. We realized a pump-probe microscopy with a 520nm pump pulse and a tunable probe pulse. The tunability on the probe arm allows us to explore better pump-probe contrast between two redox states. What's more, I will introduce my preliminary results of utilizing supercontinuum generation in a photonic crystal fiber (PCF) to realize tunability on pump wavelength. In that way, more possibilities will be unlocked. And the hyperspectral pump-probe microscope will be able to distinguish more molecules.


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label free imaging
nonlinear optics


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