Miniaturized Three-Axis Two-Photon Imaging System for Moving Mammals based on Micro-optical Systems

Abstract

This dissertation describes the design and prototyping of novel, miniaturized imaging systems intended to facilitate brain function studies in moving mammals. The imaging systems leverage optical Micro-Electro-Mechanical System (MEMS) technology to perform two-photon microscopy with cellular to sub-cellular resolution over a large field-of-view in three axes. A critical aspect of this research is to address limitations of the current state of the art in two-photon method implantable microsystems, which typically achieve less than 200 µm imaging depth range. In contrast, the systems developed in this dissertation significantly extend this range, marking a substantial advancement in the field. The implications of large working distance imaging in brain tissue are first studied with respect to MEMS scanner requirements and optical performance trade-offs. One- and two-photon imaging systems for planar (2D) imaging at large working distance are then prototyped and tested, demonstrating the adaptability and versatility of the proposed imaging systems. To extend imaging into three axes, four distinct optical designs are introduced that apply MEMS laser scanners to axial scanning in an implantable microscope. These designs include a folded-beam design for axial scanning with small, high-bandwidth mirrors; a remote scanning design for axial scanning with aberration correction; a design suitable for use with monolithic, three-axis MEMS scanners; and a design for imaging at extended depth with small diameter GRIN lenses. The overarching contribution of these designs is the integration of MEMS scanners as an axial scanning method in a miniaturized brain imaging system for the first time. These exploit the speed and cost-effectiveness of MEMS scanners, offering a significant advantage over traditional axial scanning methods in miniaturized optical imaging systems and addressing the critical need for long working distance imaging in brain imaging applications. A pivotal advancement is the ability to use the MEMS’ fast-scanning mechanism in the proposed systems. High MEMS scanning frequencies can not only capture real-time brain dynamics at about 10 frames per second but are beneficial to overcome image degradation due to the transient movement of the animal. This capability is crucial for accurate and effective imaging in live, moving mammals, providing an unprecedented level of detail and temporal resolution. The remote scanning design comprises the first miniaturized remote axial scanning method based on translational MEMS mirrors coupled with aberration correction, developed into a physical prototype. The instrument achieved a volumetric image of approximately 520 µm × 480 µm × 400 µm. Simulation results indicate diffraction-limited imaging over an extensive axial scanning range (approximately 500 µm in each axis). Imaging can be performed using MEMS scanners supporting low-frequency or static motions for beam steering, or resonant scanning to capture images in XY, XZ, or YZ planes. Initial proof-of-concept imaging studies used a knife edge test, revealing the system's capacity for achieving 1.5 µm lateral and 6 µm axial resolution. The system also proved capable of resolving minute features as small as 2µm on a standard USAF target. Further system performance evaluations included V-shaped structures and fluorescent beads. Finally, the applicability of the system was extended and confirmed through tests on mouse brain samples demonstrating the ability to image fluorescently-labeled neurons and neurites. This research reveals imaging systems can exceed existing axial scanning limitations and significantly boost neuroscience research. By making two-photon microscopy more compact and accessible, this study opens the door to crucial insights into brain health.