UC Berkeley

Understanding the complex biological mechanisms within the intact tissues requiresmonitoring biological events at subcellular or cellular spatial resolution over broad spatial coverage with sufficient temporal resolution to capture biological events of interest. Because of its ability to image deep inside the opaque biological tissues and visualize functional cells at sub-micron lateral resolution, point-scanning two-photon fluorescence microscopy (2PFM) has become one of the most common approaches for in vivo biological imaging. Here, fluorescence generation is confined to the focus of a high numerical aperture (NA) microscope objective, enabling optical sectioning deep inside living tissues. Images are generated by scanning the excitation focus across the sample and detecting emitted photons at each position. Therefore, the imaging speed and spatial coverage of 2PFM is limited by how fast the excitation focus can be scanned. The mechanical inertia of galvanometric and resonant mirrors used in conventional 2PFM limits the line scan rate to 10’s of kilohertz (kHz), resulting in 10’s-100’s two-dimensional (2D) frames per second (fps) and even lower three-dimensional (3D) volume rates. To overcome the speed and spatial coverage limit posed by the inertia nature of mechanical scanning, we developed the Free-space Angular-Chirp-Enhanced Delay (FACED) technique, a novel all-optical scanning method for 2PFM. The FACED module divides a pulsed laser into multiple beamlets with distinct temporal delays and propagation directions, achieving MHz-rate line scanning. Integrated with mechanical scanning, FACED-2PFM enables very diverse imaging modalities, including ultrafast MHz line scans, kHz 2D imaging, and large-FOV or volumetric imaging. Thus, FACED-2PFM allows for maximizing imaging spatial coverage and throughput based on the temporal characteristics of biological events. In application, FACED 2PFM achieves subcellular lateral and cellular axial resolution, imaging up to 750 µm deep in live tissue. In cerebral blood flow studies, FACED tracked over 900 vascular segments in a 1111 µm × 1000 µm FOV at 5.3 ms/frame. For voltage imaging, it captured activity from over 200 neurons in a 320 µm × 400 µm FOV, detecting subthreshold and suprathreshold signals critical for measuring neural correlation and power-law exponents in circuit models. For calcium imaging, it imaged over 14,000 neurons within a 1111 µm × 1000 2 µm × 780 µm volume, enabling comprehensive studies of orientation tuning responses across large neuronal populations. This thesis begins with an overview of imaging requirements for biological research and a discussion of conventional two-photon fluorescence microscopy principles and performance. Following this, it introduces the theoretical foundation of the FACED technique and its integration into FACED-based two-photon fluorescence microscopy. A comprehensive analysis of FACED 2PFM’s performance and capabilities is provided. The design and implementation of the FACED 2PFM hardware control system, which enables its versatile imaging modalities, and the high-speed data acquisition system, which supports sustained acquisition of vast data volumes, are then described in detail. Finally, applications of FACED 2PFM to address complex biological research challenges are presented, along with data analysis methodologies. The thesis concludes with a summary and discussion of FACED 2PFM and its potential advancements.