Abstract

Calcium imaging is widely used to monitor neuronal activity in vivo and is most commonly performed at or near the brain surface in animals expressing genetically encoded calcium indicators. However, many neural circuits of interest span multiple cortical areas and deep subcortical structures that are not readily accessible with conventional surface imaging. For deep-brain applications, light scattering in intact tissue severely degrades image quality and limits both penetration depth and spatial resolution. A common strategy to circumvent the need to image through large volumes of overlying tissue is to implant a miniature lens system (a microendoscope) directly into the brain. Although this provides optical access to deep structures, there is currently no technique that enables fast, high–signal-to-noise volumetric imaging through such lenses, forcing investigators to choose between sacrificing optical sectioning or restricting recordings to relatively modest neuronal populations. To address these limitations, we developed a novel imaging modality, RE-imaging Axial Light-sheet Microscopy (REALM), optimized for rapid three-dimensional imaging through a microendoscope. REALM employs a tilted light sheet and uses a single objective both to illuminate the sample and to collect the resulting fluorescence, thereby maintaining a compact, endoscope-compatible geometry. By combining oblique planar illumination with a re-imaging strategy, REALM achieves volumetric, cellular-resolution imaging even when using lenses with relatively low numerical aperture, as is typical in microendoscopic systems. In our implementation, the microscope collects over 40% of the light redirected by a sawtooth (blazed) mirror, compared to approximately 28% collection efficiency reported in previous work at numerical apertures that are not practical for microendoscopy. This improved photon utilization expands the feasibility of fast volumetric calcium imaging in deep-brain circuits. We demonstrate that our system can perform simultaneous multi-plane imaging in the highly scattering tissues of the mouse olfactory system and orbitofrontal cortex. Thus, REALM combines the speed and resolution advantages of light-sheet microscopy with the deep-brain access afforded by microendoscopes, enabling fast three-dimensional imaging in deep tissue.

Committee Chair

Timothy Holy

Committee Members

James Alexander John Fitzpatrick; Joseph Culver; Quing Zhu; Song Hu

Degree

Doctor of Philosophy (PhD)

Author's Department

Biomedical Engineering

Author's School

McKelvey School of Engineering

Document Type

Dissertation

Date of Award

4-29-2026

Language

English (en)

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Available for download on Tuesday, June 15, 2027

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