In this dissertation I describe the design and implementation of miniature fiber-coupled microscopes (FCMs) with active focusing for three-dimensional (3D) neuronal imaging. The goal is to provide neuroscience researchers with versatile microscopy tools to perform neuronal optical imaging of awake and mobile mice. This research is motivated by the recent advancements of powerful genetically-encoded optical proteins, including fluorescent activity sensors as well as optogenetic actuators, which permit the functional interrogation of in vivo neuronal circuits. Newly developed miniature microscope tools allow optical imaging in freely-behaving mice, but current designs do not combine optical sectioning capabilities with active focusing for full 3D-imaging.
The first three chapters in this dissertation serve as a background for current state of intravital fluorescence microscopy in neuroscience research. I argue for the importance of miniaturizing the microscope technologies that enable high-contrast imaging with optical sectioning, combined with axial focusing, to enable 3D-imaging at the rodent-scale.
I present two FCM designs that achieve full 3D-imaging using a coherent imaging fiber bundle (CIFB) for lateral imaging and an electrowetting tunable lens (EWTL) to enable electrically tunable axial focusing. The first design is a confocal FCM (C-FCM) that takes advantage of the optical sectioning capability of the CIFB to acquire high-contrast images. The second design is a two-photon FCM (2P-FCM), in which pre-compensated ultrashort pulses are propagated through the CIFB for two-photon excitation microscopy. In each section, I characterize the 3D optical performance of the FCM. Finally, as a proof-of-principle using the 2P-FCM, I show in vivo 3D-imaging of neurons and Ca 2+-activity in the motor cortex of a freely-behaving mouse.
|Advisor:||Gibson, Emily A., Restrepo, Diego|
|Commitee:||Benninger, Richard KP, Dell'Acqua, Mark L., Felsen, Gidon, Gibson, Emily A., Restrepo, Diego|
|School:||University of Colorado at Denver|
|School Location:||United States -- Colorado|
|Source:||DAI-B 79/03(E), Dissertation Abstracts International|
|Subjects:||Bioengineering, Neurosciences, Optics|
|Keywords:||Calcium imaging, Freely behaving, Miniature microscope, Neurophotonics, Optics, Two-photon|
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