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Development and application of moxifloxacin-based laser scanning microscopy

Title
Development and application of moxifloxacin-based laser scanning microscopy
Authors
이승훈
Date Issued
2020
Publisher
포항공과대학교
Abstract
Laser scanning microscopy (LSM) is a high-resolution three-dimensional (3D) imaging method based on either fluorescence or reflection. Confocal fluorescence LSM has been used for 3D cell imaging in life science with fluorescent markers, and nonlinear two-photon LSM has been used for in-vivo deep tissue imaging. These LSMs have advanced knowledge in biology. Non-invasive LSM which were either light reflection or auto-fluorescence have been used in clinical study to detect lesions in high sensitivity via cellular level examination. However, LSMs showed limited performance in clinical applications due to low-contrast and slow imaging speed. In this study, I developed new LSM methods which provide high-contrast images while still being clinically compatible. The new LSM methods used moxifloxacin, which is an antibiotic, as a cell labeling agent and provided high-contrast images of cells. Although moxifloxacin was known to have intrinsic fluorescence and to have good tissue penetration properties as a drug, its cell labeling ability was discovered by us for the first time. I characterized fluorescence properties of moxifloxacin in both linear and nonlinear excitation conditions, and developed confocal single-photon, two-photon, and three-photon microscopies using moxifloxacin. Moxifloxacin-based confocal LSM (Moxi-CLSM) is a confocal single-photon LSM method in which fluorescence is generated by single photon excitation of moxifloxacin. Moxi-CLSM had strong fluorescence comparable to commercial dye and it enabled video-rate (30 frames/second) imaging speed. Moxifloxacin-based two-photon LSM (Moxi-2LSM) is a nonlinear LSM method that produces fluorescence by two-photon excitation of moxifloxacin. Moxi-2LSM showed enhanced contrast in skin and cornea by additionally visualizing surround collagens with simultaneous second harmonic generation imaging. Moxifloxacin-based three-photon LSM (Moxi-3LSM), the another non-linear LSM methods, provided the highest contrast at deep imaging depths due to longer excitation wavelengths (> 1000 nm ) of three-photon excitation. These moxifloxacin-based LSM methods were demonstrated in several clinical applications. Moxi-LSCM could delineate malignant brain tumor by visualizing the cytoarchitecture of tumor such as densely cells and non-uniform distribution. Moxi-2LSM showed atypical aggregation and arrangement of cells in skin cancer. Keratitis pathogens were revealed by Moxi-2LSM owing to high-resolution imaging of their specific morphology like filament and elongation. In conclusion, moxifloxacin-based LSM methods could provide sub-micron (< 10-6 m) resolution in biological tissues. Their applications in clinic showed potentials as fast and sensitive surgery guiding methods or diagnosis equipment.
URI
http://postech.dcollection.net/common/orgView/200000290453
https://oasis.postech.ac.kr/handle/2014.oak/111683
Article Type
Thesis
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