THESIS
2010
xvi, 96 p. : ill. ; 30 cm
Abstract
Two-Photon Excited (TPE) fluorescence microscopy is a versatile tool in various applications. Taking advantage of TPE fluorescence microscopy using Time-Correlated Single Photon Counting (TCSPC) technique, both spectral and temporal information of the fluorescence can be collected from a three-dimensional biological specimen. In this study, we investigated the TPE fluorescence characteristics of keratins and fluorescent proteins for potential in vivo imaging of the sub-cellular structure and its function....[
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Two-Photon Excited (TPE) fluorescence microscopy is a versatile tool in various applications. Taking advantage of TPE fluorescence microscopy using Time-Correlated Single Photon Counting (TCSPC) technique, both spectral and temporal information of the fluorescence can be collected from a three-dimensional biological specimen. In this study, we investigated the TPE fluorescence characteristics of keratins and fluorescent proteins for potential in vivo imaging of the sub-cellular structure and its function.
By using a unique TPE fluorescence microscopy with multiple excitation sources, the fluorescence of three typical keratin subtypes was measured. Based on the fact that the keratin subtype profile could serve as an endogenous biomarker for cancer detection, the differences in terms of fluorescence spectral profiles and temporal decays between the keratin subtypes were identified. The results show that keratin subtypes could be separated based on the spectral and temporal differences.
Moreover, a TPE fluorescence microscopy with various pulse width excitations was designed and implemented to explore the fluorescence characteristics of the fluorescent proteins and verify the mechanism of TPE signals from the fluorescent proteins. The fluorescence dependence of mCherry and EYFP on the excitation intensity and pulse duration were both quantitatively measured. The results demonstrate that mCherry had completely different TPE fluorescence characteristics at the excitation below 760nm due to the fluorescence self-quenching from the coexisting Stimulated Emission Depletion (STED) at the excitation wavelength.
Finally, a preliminary study of a super-resolution microscopy, namely, a simplified STED microscopy was instrumented to investigate the STED in mCherry’s fluorescence at 720nm. The fluorescence inhibition by a 60ps STED pulse was observed. In addition to successfully demonstrating the feasibility of implementing STED microscopy by one laser source, the result also verified the possibility to quantify the excited state lifetime by measuring the dynamics of the depletion efficiency versus the interval between two pulses.
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