Two-photon microscopy has become a widely adopted tool for functional Calcium imaging in neuroscience research. Due to the decreased scattering at near-infrared wavelengths, two-photon excitation improves penetration depth and image contrast in the mouse brain over single-photon excitation. However, the imaging acquisition is usually performed in a laser-scanning approach, which restricts the system’s spatiotemporal bandwidth, allowing only a limited number of neurons to be captured from a 2D image plane. This dissertation focuses on the development of a single-objective lens, light-sheet excitation, two-photon microscopy approach (2P-SCAPE) that dramatically improves the system’s bandwidth over laser-scanning. The spatial multiplexing provided by light-sheet excitation resolved the trade-off between imaging speed and signal-to-noise ratio in laser-scanning. The single objective lens oblique illumination also frees up the sample space for in vivo experiments. When combined with the state-of-the-art scientific CMOS/intensified CMOS camera, 2P-SCAPE enabled high spatiotemporal bandwidth imaging of biological tissues from hundred MHz to GHz.
The first aim of the dissertation was to investigate the feasibility of two-photon light-sheet excitation given constraints such as power, signal, photodamage sources. An optimized excitation strategy was derived for laser parameters, light-sheet parameters. The performance of a near-infrared light-sheet was also investigated in a silico model. The second aim was to design and develop the 2P-SCAPE system. The imaging bandwidth and resolution of the system were improved with iterative system optimizations, including an optimized excitation strategy, dispersion management, collection throughput improvement, extended depth of focus illumination. The third aim was to apply the 2P-SCAPE system to many mouse brain and zebrafish samples for high spatiotemporal imaging of neural activities. Several spatiotemporal unmixing processing methods were applied to illustrate the rich information captured with the system. Finally, two alternative approaches to increase the penetration depth of SCAPE with NIR excitation were investigated. Proof-of-concept experiments in mouse brains also suggest they improved penetration depths over single-photon blue excitation.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/ztm9-2798 |
| Date | January 2022 |
| Creators | Yu, Hang |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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