Non-linear optical technology has become a powerful tool in biological research because it has several unique advantages over traditional optical technology. First, non-linear optical technology includes a wide variety of non-linear optical effects, such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and stimulated Raman scattering (SRS). These phenomena have distinct properties from linear optical processes, providing new opportunities to explore molecular structures and electronic states. Second, non-linear optical microscopies have advantages over traditional microscopies in many regards, such as intrinsic three-dimensional imaging capability, reduced out-of-focus photodamage and deep penetration depth. These unique properties make non-linear opt...[ Read more ]

Non-linear optical technology has become a powerful tool in biological research because it has several unique advantages over traditional optical technology. First, non-linear optical technology includes a wide variety of non-linear optical effects, such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and stimulated Raman scattering (SRS). These phenomena have distinct properties from linear optical processes, providing new opportunities to explore molecular structures and electronic states. Second, non-linear optical microscopies have advantages over traditional microscopies in many regards, such as intrinsic three-dimensional imaging capability, reduced out-of-focus photodamage and deep penetration depth. These unique properties make non-linear optical microscopies favorable for in vivo tissue imaging.

Based on the advantages of non-linear optical technology, this thesis focuses on utilizing the non-linear optical signals from large biological compounds to study biological problems at both the molecular and tissue levels. Specifically, at the molecular level, we investigated the mechanism of TPEF emission from hemoglobin, which was recently discovered, and the folding process of cytochrome c using time-resolved TPEF resonance energy transfer in a microfluidic mixer. At the tissue level, we designed a label-free multimodal non-linear optical microscope to study normal and abnormal skeletal muscle development using non-linear optical signals, including the TPEF of tryptophan in proteins and reduced nicotinamide adenine dinucleotide in mitochondria, the SRS of C-H bonds in proteins and lipids, and the SHG of myosin in the sarcomere. We also integrated femtosecond laser surgery system into our non-linear optical microscope to perform laser ablation of biological tissues. We found that the femtosecond laser can create fluorescent compounds in biological samples after laser surgery via a non-linear process. The mechanism of creating such fluorescent compounds was also explored, along with their molecular structures. Finally, the potential applications of such laser produced fluorescent compounds were demonstrated in the mouse brain and muscle tissue in vivo.