UC Davis

Optical methods provide powerful means to probe living biological systems with high spatial and temporal precision. Light-tissue interactions alter the intensity, phase, or polarization of incident light, or produce new wavelengths (e.g., fluorescence, Raman scattering) that differ from the original light. By analyzing these changes or newly generated emissions, researchers can uncover structural and functional properties of biological samples. For in-vivo applications, achieving deep tissue penetration while preserving high spatial resolution is especially valuable. Near-infrared (NIR) light in the 650–1350 nm range is optimal for this purpose, as it has low water absorption and reduced scattering compared to visible light, enabling deeper tissue penetration. This thesis focuses on the development of advanced optical tools for in-vivo bio-sensing and bio-imaging using NIR light. We present two optical modalities for distinct applications: interferometric near-infrared spectroscopy (iNIRS) for transabdominal fetal oximetry, and miniaturized two-photon microscope for recording neuronal activity in freely-behaving mice. For the miniaturized two-photon microscope, we present two optical systems including a time-multiplexed version which can support high-speed imaging, and a millimeter-scale field-of-view version which can image a large-scale neuronal circuit. In bio-optical sensing, we present an innovative approach to measure fetal oxygenation transabdominally using interferometric near-infrared spectroscopy. Assessing fetal oxygenation is crucial for evaluating fetal health during labor, and NIRS offers a non-invasive method for this purpose. Traditional NIRS techniques determine biological properties by quantifying chromophore concentrations based on light attenuation or temporal phase variations. Here, we introduce iNIRS, a time-domain (TD) NIRS technique optimized for transabdominal fetal oximetry. Unlike continuous-wave (CW) NIRS, the iNIRS approach provides significantly enhanced sensitivity to deep tissues by leveraging time-resolved reflectance, enabling separation between signals originating from shallow and deep tissue layers. In transabdominal fetal oximetry, separating fetal signals from maternal signals is essential. The iNIRS method addresses this by directing light at the maternal abdomen, where it penetrates both maternal and fetal tissues before reaching the detector. Photons that reach the fetal layer scatter back toward the surface and are detected as a mixed signal combining maternal and fetal contributions. This deep-tissue signal can be differentiated from maternal surface signals using time-resolved reflectance. Further separation of the fetal signal from the mixed signal is achieved with adaptive noise cancellation. We successfully demonstrated the extraction of fetal heart rate in a pregnant sheep model. By placing a compact optical probe on the abdomen of pregnant sheep, we could detect fetal signals from depths ~1.6 cm within the maternal uterus. Together, these advancements position iNIRS as a promising tool for real-time, non-invasive detection of fetal hypoxemia and comprehensive assessment of fetal health during labor. In bio-optical imaging, we developed an innovative miniaturized two-photon microscope using near-infrared femtosecond lasers to image neural activity in freely-behaving animals. Two-photon calcium imaging enables the simultaneous readout of neural activity with cellular resolution in deep tissues; however, such systems have traditionally been restricted to benchtop settings. While head-fixed setups and virtual reality environments provide insights into neuronal circuits underlying behavior, they still limit animals to a controlled set of behaviors. Consequently, naturalistic behaviors, such as social interactions (requiring multiple subjects) or unrestricted 2D navigation (requiring full head movement), cannot be fully explored in these settings. The MINI2P overcomes these limitations by reducing the size and weight of the microscope, enabling it to be mounted or implanted directly on the animal's head. However, designing optical imaging systems faces inherent trade-offs in FOV, resolution, and frame rate. Physical constraints in lens design, device dimensions, and scanner capabilities make it challenging to optimize all three parameters simultaneously within a single system. This work proposes two novel configurations for a miniaturized two-photon microscope that address the trade-offs between frame rate, resolution, and FOV. Time-multiplexed miniaturized two-photon microscope (TM-MINI2P) is designed to increase frame rate without compromising high resolution, enabling the capture of neural activity across multiple planes. This design allows for expanded imaging in the z-axis, capturing the neuron firing simultaneously across three dimensions. The millimeter miniaturized two-photon microscope (MM-MINI2P) expands the FOV to nearly the millimeter scale while maintaining high resolution (NA = 0.337) in a compact design. Existing MINI2P systems typically image a 500×500 μm2 FOV with high resolution (NA = 0.3); the proposed system offers a similar resolution but with an almost four times expanded FOV. Both TM-MINI2P and MM-MINI2P are well-suited for studying extensive neural activity in freely moving animals, thus broadening the applications of two-photon imaging in neuroscience.