THESIS
2017
xx, 164 pages : color illustrations ; 30 cm
Abstract
Nonlinear optical (NLO) microscopy is the current method of choice for deep-tissue imaging.
Compared to conventional 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 t...[
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Nonlinear optical (NLO) microscopy is the current method of choice for deep-tissue imaging.
Compared to conventional 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.
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