Examination Committee

Prof Zhigang LI, MAE/HKUST (Chairperson)
Prof Jianan QU, ECE/HKUST (Thesis Supervisor)
Prof Shaoqun ZENG, College of Life Science and Technology, Huazhong University of Science and Technology (External Examiner)
Prof Bertram SHI, ECE/HKUST
Prof Kevin CHEN, ECE/HKUST
Prof Tom CHEUNG, LIFS/HKUST
 

Abstract

Nonlinear optical (NLO) microscopy is the current method of choice for deep-tissue imaging. Compared to traditional microscopies, the advantages of NLO technology include its inherent three-dimensional imaging capability, deep penetration depth and less out-of-focus photo-damage. The multiple modalities of NLO microscopy, such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and stimulated Raman scattering (SRS), provide unique contrast mechanism to probe a variety of endogenous molecules in biological specimens. Integrated with advanced spectral and time-resolved fluorescence detection technology, the NLO microscopy enables structural and functional imaging of biological tissues and processes. These unique properties make NLO microscopy a powerful tool for in vivo tissue imaging. 
 
This thesis focuses on utilizing the NLO spectroscopy and imaging techniques to study biological tissues and processes. Specifically, we develop an infrared laser heat shock microscope system to study the fate mapping of microglia in zebrafish. With a newly developed transgenic zebrafish model, we achieve precise control of the transgene expression through localized laser-induced heat stress. This enables us to label specific cells in a confined anatomy with high spatial-temporal resolution. To achieve high-efficiency single-cell labeling, we develop a state-of-art two-photon fluorescent thermometry to measure the local temperature rise induced by laser heat shock in zebrafish tissues in vivo. Based on the temperature measurement result, we can finely control the infrared laser power and achieve single-cell labeling in a variety of cell types in zebrafish. In addition to the single-cell labeling and cell fate mapping study, we design a multimodal NLO microscope to investigate the morphological and functional characteristics of different types of mouse tissues. First, we achieve label-free imaging of the multi-layer structure of mouse retina. TPEF and SRS imaging reveal the detailed cellular structure of retinal ganglion cells, the most important sensory neurons in retina. Moreover, multiple endogenous fluorophores in retinal pigment epithelium are differentiated based on spectral and time-resolved fluorescence analysis. Second, we use the multimodal NLO microscope to study the cartilage development in mouse. Through the texture analysis of collagen fibrils and morphological imaging of chondrocytes, we identify the significant role of a motor protein, Kif5b, in the development of chondrocytes and extracellular matrix in mouse cartilage. Lastly, we use the intrinsic fluorescence of coenzymes to study the thermogenic properties of adipose tissues in mouse in vivo. Subgroups of brown adipocytes with different metabolic characteristics are identified through NLO imaging. Furthermore, a fiber-based spectroscope system is developed to measure the optical redox ratio (ORR) in deep adipose tissues. Rapid and intensive thermogenic process is successful recorded based on ORR measurement in different types of adipocytes in vivo.