Investigation of Roles of 1O2 in Photochemistry of an A-D System and Development of Three-photon Induced Fluorescence Microscopy

Abstract

This dissertation covers two research topics. The first topics examines the role of singlet oxygen in the photochemistry of the A-D system. The second topic presents the development and application of three-photon induced fluorescence microscopy. In the first part, background on the photochemistry, formation of radical ion pairs and exciplex, magnetic field effect on radical ion pairs mechanism, the significance of singlet oxygen, and three-photon induced excitation microscopy is presented briefly. In the second part, the photodegradation process of pyrene-(CH2)12-O-(CH2)2-N,N-dimethylaniline (Py-DMA), serving as a model molecular system for exciplex-forming A-D systems, is meticulously examined in solution. The alkyl chain-linker ensures efficient electron transfer between Py and DMA, enabling exciplex formation at concentrations as low as ~5 μм, free from the interferences dominant in solid-state devices (domain-electrode interface, domain morphological change, accumulation of defects, and so on). The photodegradation mechanism of Py-DMA is proposed for the first time based on chemical identification using steady-state spectroscopy and LC-UV-MS techniques. The mechanism predicts Py-MMA (N-monomethylaniline) and Py-MFA (Nmethylformanilide) as primary products and is verified by crosschecking experimental data from FT-IR and 1H NMR, as well as quantum mechanical calculation data. The heavy involvement of molecular oxygen (O2) predicted in the mechanism is confirmed by a series of deoxygenated condition experiments. Although we focus on the two primary photodegradation products, secondary, tertiary, and subsequent photodegradation products are also reported, such as PyOH-MPCA (methylphenylcarbamic acid), Py-FA (formanilide), and even unspecified black carbon precipitates. With recent emerging evidence of a close correlation between the stabilities of optoelectronic devices and their active molecules, the molecular photodegradation pathways of Py-DMA will shed light on the molecular design for exciplex-based optoelectronic devices with longer lifespans. In the third part, singlet oxygen (1O2), one of the reactive oxygen species known for its strong reactivity underscores the importance of controlling the amount of singlet oxygen produced. Here, we observe optically the magnetic field effect (MFE) on singlet oxygen generation using pyrene-(CH2)12-O-(CH2)2-N,N-dimethylaniline ii (Py-DMA) as a A-D-based photosensitizer. The photosensitization reaction between the Py-DMA and oxygen molecule reached saturation under magnetic field of approximately 100 mT. This field led to a negative MFE of approximately 5% for singlet oxygen generation from triplet-state radical ion pair, while a positive MFE of approximately 13% observed from exciplex emission. We anticipate that this research will offer valuable insights for the development of A-D-based photosensitizers. In the fourth part, we present the development and application of a three-photon induced fluorescence (3PIF) microscopy method for three-dimensional (3D) imaging of live HeLa cells. Leveraging the intrinsic fluorescence properties of tryptophan (Trp), this technique enables cell imaging without the need for staining or genetic engineering. To verify the source of fluorescence, glutathione S-transferase (GST) protein and its mutant form, where all Trp residues were replaced with alanine, were utilized. The average fluorescence lifetime of Trp in both HeLa cells and Trp solution was measured using time-correlated single-photon counting (TCSPC), and fluorescence intensity images were converted into Trp concentration maps. Using these 3D Trp distribution data, we calculated various biological parameters for HeLa cells. The mean concentration of Trp in a single HeLa cell was determined to be 4.0 0.9 mM. Cell volume, estimated by counting the number of pixels in the cell image, was found to be 4500  1000 m3 , consistent with previously reported values. From these measurements, the average amount of Trp per HeLa cell was estimated to be 18.6  7 fmol. Our results demonstrate that 3PIF microscopy is a powerful tool for non-invasive, three-dimensional imaging of Trp in live cells, providing valuable insight into cellular structures and functions. In the fifth part, the setup of a three-photon induced fluorescence microscope and LabVIEW code for operating microscope are reviewed.