Fluorescence microscopy has played a pivotal role in the realm of biological and biomedical research, allowing researchers to delve into the intricacies of living organisms at the cellular and molecular levels. By using fluorescent probes, one can visualize specific molecules and structures within cells, fundamentally transforming our comprehension of biology and medicine. However, fluorescence microscopy faces its own set of challenges, namely, photobleaching and photodamage. Photobleaching involves the irreversible loss of fluorescence signal during imaging, while photodamage results in harmful effects on cells. Both severely limit fluorescence signal and observation time. Although remedies exist to mitigate these problems, most of them rely on chemical approaches. In this dissertation, to address these issues, I investigated two optical approaches that exploit control of light either in space or time.
Firstly, I developed multiline scanning confocal microscopy (mLS) with a digital micro-mirror device. This method provides programmable patterns of the illumination beam as well as the detection slit. Through experimental results and optical simulations, I assessed the depth discrimination of mLS under different optical parameters and compared it with a multipoint system such as spinning disk confocal microscopy (SDCM). Surprisingly, under the same illumination duty cycle, I found that mLS offers better optical sectioning than SDCM. Importantly, the parallelized line illumination showed a much lower photobleaching rate compared to single-line scanning microscopy, while their optical sectioning capabilities remained similar. I applied this technique to visualize heterogeneous mouse epiblast stem cells, a challenging task in imaging.
Secondly, I delved into low photobleaching rate two-photon microscopy (2PM). 2PM inherently provides excellent optical sectioning due to its nonlinear effects, making it suitable for high-resolution imaging within biological tissues. However, the high peak power of ultrafast pulses has always been associated with severe photobleaching, posing a longstanding challenge. I found that controlling the repetition rates of ultrafast lasers is a potential strategy to enhance photostability. Specifically, I used repetition rates lower or higher than 80 MHz in 2PM and conducted systematic experiments to investigate how optical parameters such as wavelengths, excitation powers, and pulse schemes can influence the photobleaching kinetics of fluorescent proteins and organic fluorophores. This thesis embarks on a journey to explore innovative strategies and methodologies aimed at reducing photobleaching while maintaining high-quality imaging in the realm of fluorescence microscopy.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2023-1008 |
| Date | 01 January 2023 |
| Creators | Weng, Chun-Hung |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Thesis and Dissertation 2023-2024 |
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