UC Berkeley

Two-photon fluorescence microscopy (2PFM) is a superior alternative to conventional fluorescence microscopy with its higher contrast and deeper tissue penetration. It has become a method of choice for biological imaging of complex samples, such as the mammalian brain. For high-resolution imaging, however, 2PFM suffers from optical aberrations induced by the sample and imaging system, which lead to degraded image quality. Adaptive optics (AO) has been applied to 2PFM to remove these aberrations, hence improving image resolution and enhancing image contrast.

This thesis explores two applications of AO in 2PFM to achieve high-precision imaging of neurons in the mouse brain.

The first application is divergence-based remote focusing (Chapter 2). Capable of imaging through scattering and opaque tissues, 2PFM has been widely applied to three-dimensional (3D) imaging of biological samples. Remote focusing by controlling the divergence of excitation light is a common approach to scan the focus axially. However, changing the beam divergence induces distortion to the excitation wavefront, which leads to an aberrated focus and degraded image quality away from the natural focal plane. To maintain the focal quality, we used indirect-wavefront-sensing-based AO to measure and correct divergence-induced aberrations and achieved diffraction-limited resolution during remote focusing.

The second application is in vivo mouse retinal imaging (Chapter 3). The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. To characterize and correct mouse ocular aberration, we applied direct-wavefront-sensing-based AO to in vivo two-photon imaging of the mouse retina. We demonstrated the capability of AO-2PFM in longitudinally tracking structural and functional changes of the mouse retina with high accuracy. Our method represents an important advancement in microscopic investigation of retinal pathology and pharmacology in vivo for disease diagnosis and treatment.

Besides technological developments of AO, this thesis also presents the applications of 2PFM in functional studies of mouse primary visual cortex (V1) and visual thalamus (Chapter 4) with in vivo calcium imaging. Measuring the neuronal activities of V1 neurons and their thalamocortical afferents in response to visual stimuli, we investigated the spatial organization of orientation tuning in V1 and found significant tuning similarities among nearby neurons in both L2/3 and L4 of V1.