UC Berkeley

A microscope with optical sectioning capability allows for the acquisition of a clear image from a thin slice within a thick sample. In contrast, optical microscopy without such capability has out-of-focus light blurring in-focus details when imaging thick samples, degrading contrast and resolution. For this reason, microscopy modalities with optical sectioning are essential tools for tissue imaging, enabling the acquisition of structural and functional information at high resolution and precision. Various approaches to achieving optical sectioning have been developed, each suited to different application scenarios. This thesis aims to advance the capability and explore the application of three optical microscopy modalities: confocal fluorescence microscopy, three-photon fluorescence (3PF) microscopy and third-harmonic generation (THG) microscopy, categorized into linear and nonlinear imaging, respectively, with a focus on their use in live biological systems. Chapter 1 briefly introduces the imaging modalities involved, the challenges faced by each technology, and the approaches we are employing to address these challenges.In Chapter 2, this thesis incorporates a novel adaptive optics (AO) approach based on frequency-multiplexed aberration measurement into single-photon confocal fluorescence microscopy. Confocal microscopy achieves optical sectioning by using a confocal pinhole to reject out-of-focus fluorescence light. However, it can be affected by optical aberrations induced by imperfect optics and complex samples, which degrade imaging quality. By correcting optical aberrations affecting both the excitation and emission light with our AO approach, we demonstrate that this method can measure aberrations using signals from features of any size and substantially improve the image quality in both nonbiological and biological samples. In Chapter 3, this thesis explores the application of combined 3PF and THG microscopy for plant root imaging and shows how AO THG microscopy improves image quality in various living systems. Both 3PF and THG imaging modalities achieve optical sectioning by employing a nonlinear process that generates a signal confined to the focal plane. While the 3PF signal originates from fluorophores, the THG signal arises from inherent local optical heterogeneities, eliminating the need for fluorescent labeling. Combined with the deep penetration capability enabled by near-infrared excitation, we demonstrate in situ imaging of live roots and microbes at high spatiotemporal resolution, revealing key plant root structures from the surface to deep within the root. Furthermore, we apply the aforementioned AO approach to THG microscopy, achieving high-resolution in vivo imaging within various biological systems. In Chapter 4, this thesis describes ongoing efforts to develop a homodyne THG microscopy system. To address the challenge of low optical conversion efficiency in THG imaging of biological samples, a homodyne process that employs the coherent mixing of the sample-generated THG signal with a reference THG signal is used to enhance the conversion efficiency. Our home-built THG homodyne microscopy aims to enable long-term imaging of living systems with minimal photodamage, with a specific focus on studies of delicate root structures. Together, this thesis presents several advancements in optical microscopy techniques and aspires to enable future studies using these improved tools in various live biological systems.