Coded computational illumination and detection for three-dimensional fluorescence microscopy
- In vivo calcium imaging enables the optical monitoring of neural activity at the level of individual neurons in real time, necessitating the development of high speed, three-dimensional (3D) fluorescence microscopy techniques with at least single-neuron spatial resolution. Because a typical widefield microscope intrinsically produces only two-dimensional images, various illumination and detection coding strategies have been implemented to address the challenge of 3D fluorescence microscopy, utilizing either precisely structured and temporally scanned illumination patterns, such as in two-photon laser scanning microscopy or coding of the emission, as in light field microscopy, respectively. However, many single-focal illumination coding strategies have limited acquisition speeds, while detection-coding-only strategies requiring computational reconstruction of the 3D volume are limited by optical aberrations of the tissue. We present a 3D calcium imaging approach utilizing both multifocal scanned two-photon laser excitation for illumination coding and detection coding with the light field microscopy approach suitable for in vivo mammalian calcium imaging. A holographic 3D multifocal illumination pattern is targeted only towards pre-localized neurons avoiding the unnecessary illumination of other regions. The resulting fluorescence emission is coded and detected on an image sensor and deconvolution is used to recover the neural activity at each site. We present the design and optimization of such an imaging strategy, and validate the approach with experimental measurements. Finally, we demonstrate the application of this approach to in vivo mouse calcium imaging. The design and implementation of another technique, frame-projected independent fiber photometry, enabling the optical recording and control of neural activity in freely moving mammals with region-level spatial resolution, is presented in a dedicated chapter as well, including simultaneous recording from multiple brain regions in a mouse during social behavior, two-color activity recording, and optical optogenetic stimulation eliciting dynamics matching naturally observed patterns.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Yang, Samuel J
|Stanford University, Department of Electrical Engineering.
|Statement of responsibility
|Samuel J. Yang.
|Submitted to the Department of Electrical Engineering.
|Thesis (Ph.D.)--Stanford University, 2016.
- © 2016 by Samuel Yang
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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