UC Berkeley

Brain is composed of complex neural networks that work in concert to underlie the animal’s cognition and behavior. Optical microscopy has become an indispensable tool to study brain due to its non-invasiveness and high spatial resolution. Two-photon fluorescence microscopy is the method of choice to image the optically opaque mammalian brain. However, conventional two-photon fluorescence microscopy has to perform serial 3D point scanning for volumetric imaging, which renders it extremely difficult to study neural circuits in 3D at subcellular resolution with sufficient imaging speed.Taking advantage of the fact that neurons remain largely stationary during in vivo imaging, when their temporal activities are the subject of interest, one can acquire an axially projected view of the objects without constantly tracking their 3D location. Incorporating a Bessel-like beam into a conventional two-photon fluorescence microscope extends the system’s depth-of-focus, which turns the 2D frame rate into an axially projected volume rate. As a result, Bessel focus scanning technology enables high-speed volumetric imaging without sacrificing the lateral resolution and reduces data size by an order of magnitude when compared with conventional serial 3D scanning. This thesis first explores the application of Bessel focus scanning technology in two-photon fluorescence microendoscopy to achieve high-throughput neural circuit imaging in deeply-buried nuclei of the mouse brain. The thesis then presents efficient data analysis approaches for high-throughput calcium neural imaging data. Finally, the thesis depicts a custom-designed and easy-to-operate two-photon fluorescence microscope combining several state-of-the-art technologies together, i.e., adaptive optics, Bessel focus scanning technology, and remote focusing, which will open up possibilities for neurobiology questions that could not be well addressed before.