Development of Adaptive Optics Multiphoton Microscopy for Brain Research

Abstract

Imaging the tissues of live animals is critical for biological study, as it provides direct observation of function and mechanisms in their natural physiological state. Multiphoton microscopy (MPM), a technology that offers significant advantages over traditional optical microscopy for deep tissue in vivo imaging, has greatly advanced research in biology and disease. Benefiting from the physical property of nonlinear excitation, MPM possesses intrinsic sectioning ability, enabling the acquisition of three-dimensional (3D) volumetric images through tissue. Additionally, the longer wavelength of the excitation light required by MPM results in reduced scattering when traversing tissue, allowing more unscattered photons to form the excitation focus at deeper tissue layers. Furthermore, the well-developed fluorescent proteins, harmonic generation, and autofluorescence provide a wide range of highly specific labelling options for the structures of interest, facilitating their direct use in MPM for multimodal imaging. Despite the achievements of MPM in recent years, acquiring high-resolution images at deep tissue in a non-invasive manner remains challenging, as scattering and aberration induced by the opaque thick tissue significantly distort the light.

Given that MPM is a powerful yet imperfect imaging technology, my thesis work focuses on enhancing its performance and extending its application for imaging the function and structure of biological tissues that are otherwise inaccessible without my developed technology. Specifically, we first constructed a three-photon microscope with adjustable dispersion compensation and multimodal imaging capabilities to study neuron and blood vessel structures throughout the entire mouse cortex. Next, we developed an advanced adaptive optics (AO) technology named analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS), which enables the recovery of diffraction-limited resolution through precise measurement and correction of aberration and scattering. Additionally, we implemented ALPHA-FSS on the three-photon microscope and combined it with conjugate AO remote focusing (CAORF) for large field of view (FOV) aberration correction, achieving non-invasive sub-cellular imaging up to 750 µm through an intact mouse skull. Furthermore, we advanced ALPHA-FSS to multiplexing digital focus sensing and shaping (MD-FSS) for two-photon high-resolution imaging of the brain in awake, behaving mice through a thinned skull and optical clearing window, which is challenging due to motion artifacts in awake animals. MD-FSS comprises two major innovations: beam multiplexing with phase encoding and digital phase decoding, which significantly accelerates the speed of AO to approximately 0.1 seconds per measurement.

Utilizing this novel rapid AO technology, we achieved near non-invasive brain imaging of the mouse cortex at high resolution in awake, behaving mice and discovered clear differences in cell morphology and function between awake and anesthetized states, underscoring the necessity of imaging awake animals. Finally, we extended the application of single beam digital focus sensing and shaping (SD-FSS), developed alongside MD-FSS for aberration correction in holographic patterns, and introduced a new technology of adaptive optics holography. In this process, we first measured the aberration using SD-FSS and then incorporated aberration correction into the algorithm of phase wavefront calculation for holography. This aberration-corrected phase wavefront is effective in generating diffraction-limited holography pattern through the mouse skull. With AO-assisted holography generation, we demonstrated multi-target photobleaching with high accuracy and efficiency, indicating that AO holography technology is effective for precise cell manipulation.

Date of Award	2026
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Jianan QU (Supervisor)