For centuries, optical microscopy has greatly facilitated our observation and understanding of microcosm. The innovation of optical microscopy technology has been proven to be the driving force behind the development of biology and medicine. In the past two decades, much effector has been devoted to the development of nonlinear optical (NLO) microscope based on different kinds of NLO contrast mechanisms. The NLO microscope provides a range of unprecedented capabilities, including larger penetration depth, inherent 3-D optical sectioning and less photo-toxicity, etc. Its application in bio-related research has been widely explored. In particular, intrinsic NLO signals of two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), third harmonic generation (THG), coherent...[ Read more ]

For centuries, optical microscopy has greatly facilitated our observation and understanding of microcosm. The innovation of optical microscopy technology has been proven to be the driving force behind the development of biology and medicine. In the past two decades, much effector has been devoted to the development of nonlinear optical (NLO) microscope based on different kinds of NLO contrast mechanisms. The NLO microscope provides a range of unprecedented capabilities, including larger penetration depth, inherent 3-D optical sectioning and less photo-toxicity, etc. Its application in bio-related research has been widely explored. In particular, intrinsic NLO signals of two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), third harmonic generation (THG), coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), have emerged as popular contrast mechanisms for the imaging of a variety of biomedical specimens in vivo.

This Ph.D. research focuses on developing label free and multimodal NLO microscopy and exploring its biological and biomedical applications. First we home built a TPEF microscope that can provide simultaneous time- and spectral-resolved detection capabilities. This is the foundation for more advanced multimodal NLO microscopy. Then we extended the excitation wavelength range of the standard Ti:sapphire femtosecond laser by incorporating supercontinuum light generated from photonic crystal fiber. This allows the construction of a multi-color excitation two-photon microscope, which can simultaneously acquire the TPEF signals of multiple fluorophores and SHG signal of collagen. Moreover, using the spectrally extended excitation source, we discovered, for the first time, that hemoglobin emits strong Soret band fluorescence, when it is two-photon excited by the visible light. This enables TPEF microscopy to become a powerful tool for in vivo label-free imaging of microvasculature in tissues. Furthermore, we developed a multimodal NLO microscope that integrates the multiplex CARS (M-CARS), TPEF and SHG together. In our M-CARS measurement, the nonresonant background can be effectively reduced by a simple subtraction method. Finally, the system is upgraded to SRS based multimodal microscope integrating the SRS of CH₃ stretching, TPEF of tryptophan and Flavoproteins, sum frequency generation (SFG) of collagen and THG into a single platform. At the same time, we explore the biomedical application opportunities of each kind of these NLO microscopy techniques.