The application of light in biological research has significantly enhanced our understanding of the complex processes within living organisms, resulting in a multitude of breakthroughs and advancements in fields such as medicine and biotechnology. However, one significant challenge that limits the utility of light in vivo lies in the strong absorption and scattering of photons by the biological tissues, which prevent efficient and precise light delivery deep into the body. To address this challenge, we developed acoustic and optical bio-interfaces that can non-invasively deliver photons deep inside the tissue for various applications. First, we synthesized multi-color mechanoluminescent (ML) colloids as ultrasound-mediated nanoscopic light sources using a biomineral-inspired suppressed dissolution approach. This synthesis approach utilizes a unique phenomenon of suppressed dissolution observed in Nature, and can produce ML colloids down to 20 nm from their micro-sized precursors while preserving the optical properties. The produced ML colloids can be systemically delivered into the blood stream, and produce transient and localized light emission upon the stimulation of deep-penetrating focused ultrasound (FUS). We demonstrated that the ultrasound-mediated light emission can activate channelrhodopsin-2 (ChR2)-expressing neurons in the mouse brain, producing significant behavioral and histological changes. The use of an acoustic interface eliminates any brain implants or scalp incision, thus allowing non-invasive neuromodulation. Second, using a similar synthesis strategy, we produced multi-color persistent luminescence (PerL) colloids as circulation-delivered light sources. These PerL colloids can store photoexcitation energy in the lattice, and gradually release it as light in an extended period of time. We showed that these PerL colloids are among the brightest afterglow materials ever reported, with short emission wavelengths desired for activating light-sensitive proteins. We then demonstrated the utility of these PerL colloids in excitation-free imaging of brain vasculatures after systemic delivery, and used them as internal light sources to excite endogenously expressed fluorescent proteins in the mouse brain. Third, we developed an optical neuromodulation technique in the second near-infrared window (NIR-II, 1000−1700 nm) using semiconducting polymer nanotransducers with bandgap engineering. After local delivery into the brain, these nanotransducers strongly absorb brain-penetrant NIR-II light and efficiently convert it into heat, which can then activate ectopically expressed temperature sensitive ion channels for neuromodulation. We demonstrated the utility of this NIR-II neuromodulation technique in the motor cortex, hippocampus, and ventral tegmental area (VTA) of mice, producing significant behavioral, histological, and electrophysiological changes. The use of an NIR-II interface allows complete elimination of invasive brain implants and head tethering, which will be advantageous for the future study of social interactions in rodents.

Imaging with high spatial resolution and high specificity within intact tissues at depth has long been a critical research objective for implementation in biological studies. The development of imaging tools with the capability of deep imaging at cellular resolution would allow for more realistic and complicated biological hypotheses to be tested in their natural environment - intravitally. The most challenging aspects of such tool developments involve light scattering and aberration, which cause the light to distort along its propagation direction, limiting both its imaging depth and resolution. This thesis attempts to provide several solutions to overcomes these challenges. To overcome light scattering, imaging within the short-wave infrared region (SWIR, wavelength 1 - 2.5 micrometers) is explored in chapters 2-4. In chapter 2 and 3, reflectance confocal and fluorescence confocal microscopy are demonstrated providing 2-4 times deeper penetration than any previously reported work and preclude the possibility of using one-photon confocal microscopy for deep imaging, a method that has been rarely discussed. Furthermore, a study on the impact of staining inhomogeneity on the depth limit of fluorescence confocal microscopy also demonstrated the potential of confocal microscopy combined with SWIR and low staining inhomogeneity to achieve unprecedented imaging depth. After demonstrating the deep imaging capability of one-photon imaging at depth with a SWIR light source, a multimodal system combining three-photon, third-harmonic, and optical coherence microscopy (OCM) is demonstrated in chapter 4. This multimodal system was able to achieve simultaneous imaging depth comparable to imaging with multiple contrast mechanisms in terms of the fluorescence, the harmonic signal, and the backscattering. Furthermore, this multimodal system provided complementary information about the mouse in vivo and represented a powerful intravital biological imaging tool. To overcome light aberration, adaptive optical methods are demonstrated in chapters 5-7. In chapter 5, a sensorless, adaptive optics, and indirect wavefront sensing system is demonstrated to improve SWIR-excited three-photon imaging, achieving about 7x signal enhancement in the mouse hippocampus area. This method is based on using the nonlinear three-photon fluorescence signal as feedback and involves light exposure during the optimization process. To reduce light exposure, a more direct wavefront sensing method is explored using a SWIR OCM system to directly sense the complex field of a biological sample in chapter 6. The advantage of this system, including its potential high-speed wavefront sensing and offline wavefront estimation, and its limitations with respect to phase stability are discussed. Finally, in chapter 7, a direct wavefront sensing method based on a cheap silicon wavefront sensor is presented. This method provides a convenient approach for aberration measurement with any experiment that involves SWIR ultrafast laser. This thesis shows the great promise to achieve high-resolution deep tissue imaging at a larger depth by combining longer wavelength at short-wave infrared region and adaptive optics. It is anticipated that this thesis work will open doors to much more exciting biological research in the near future.