Design and Development of Multiscale Microscopic Imaging Systems for Neuroscience Applications

Multiscale imaging systems enable the measurement of micro-scale activity from individual neurons and mesoscale activity from diverse brain regions for understanding functional properties and correlations of widely separated neurons. However, these techniques are often expensive, with complex designs limiting extensive neuroscience research. The main goal of this thesis research is to develop two cost-effective in vivo fluorescence imaging systems to visualize structural and functional details of the brain in rodents. We designed and developed a two-photon microscope (2PM) utilizing a custom-built fiber optic laser to deliver the short intense pulses required for two-photon excitation. We have obtained high-resolution images from in vitro samples from a field of view (FOV) of 90 x 90 μm using the 2PM. Using low repetition rate lasers for two-photon imaging demonstrated negligible heating in tissue-mimicking phantom, highlighting the importance of similar excitation sources. The 2PM was converted into a further advanced three-photon microscope utilizing an excitation wavelength of 1300 nm. In a depth comparison study using brain sections, three-photon microscopy showed capability for deep tissue imaging compared to two-photon imaging. We have also designed and developed a mesoscope for in vivo imaging. Mesoscope configuration with large FOV of 12.6 x 10.5 mm provided a spatial resolution of 4.92 μm and the reverse configuration with a smaller FOV of 6 x 5 mm provided a resolution of 2.46 μm. Finally, both the microscopes were utilized for in vivo imaging in rodents. The large FOV configuration of mesoscope showed capability for recording calcium activity from the entire cortical region of a rat brain. The reverse configuration was identified to be suitable for investigating localized brain activity from a mouse brain cortex. In vivo imaging using 2PM compared the efficiency of two common biosensors RGECO and GCaMP when used with an excitation wavelength of 1030 nm. The fluorescence detection using GCaMP showed improved efficiency compared to RGECO. Both microscopes are designed with cost-effective and highly efficient elements for maximum fluorescence detection and design flexibility for future developments. In forthcoming studies, these technologies will gain insights into auditory dysfunction in mouse models of Autism Spectrum Disorder.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Physics

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2022

Author dc:creator

• Jose, Ashly

Advisors dc:contributor.advisor

• Vanholsbeeck, Frederique
• Cheyne, Juliette
• Broderick, Neil

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/64492

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/64492