Date of Award

August 2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Engineering

First Advisor

Ramin Pashaie

Committee Members

Brian S R Armstrong, Chiu Tai Law, Carol J. Hirschmugl, Taly Gilat Schmidt

Keywords

Fluorescent Protein, Monte Carlo, Optical Imaging, Optical Phase Conjugation, Optogenetics, Tissue Optical Properties

Abstract

Study of the brain microcircuits using optogenetics is an active area of research. This method has a few advantages over the conventional electrical stimulation including the bi-directional control of neural activity, and more importantly, specificity in neuromodulation. The first step in all optogenetic experiments is to express certain light sensitive ion channels/pumps in the target cell population and then confirm the proper expression of these proteins before running any experiment. Fluorescent bio-markers, such as green fluorescent protein (GFP), have been used for this purpose and co-expressed in the same cell population. The fluorescent signal from such proteins provides a monitory mechanism to evaluate the expression of optogenetic opsins over time. The conventional method to confirm the success in gene delivery is to sacrifice the animal, retract and slice the brain tissue, and image the corresponding slices using a fluorescent microscope. Obviously, determining the level of expression over time without sacrificing the animal is highly desirable. Also, optogenetics can be combined with cell-type specific optical recording of neural activity for example by imaging the fluorescent signal of genetically encoded calcium indicators.

One challenging step in any optogenetic experiment is delivering adequate amount of light to target areas for proper stimulation of light sensitive proteins. Delivering sufficient light density to a target area while minimizing the off-target stimulation requires a precise estimation of the light distribution in the tissue. Having a good estimation of the tissue optical properties is necessary for predicting the distribution of light in any turbid medium. The first objective of this project was the design and development of a high resolution optoelectronic device to extract optical properties of rats' brain tissue (including the absorption coefficient, scattering coefficient, and anisotropy factor) for three different wavelengths: 405nm, 532nm and 635nm and three different cuts: transverse, sagittal, and coronal. The database of the extracted optical properties was linked to a 3D Monte Carlo simulation software to predict the light distribution for different light source configurations. This database was then used in the next phase of the project and in the development of a fluorescent tomography scanner.

Based on the importance of the fluorescent imaging in optogenetics, another objective of this project was to design a fluorescence tomography system to confirm the expression of the light sensitive proteins and optically recording neural activity using calcium indicators none or minimally invasively. The method of fluorescence laminar optical tomography (FLOT) has been used successfully in imaging superficial areas up to 2mm deep inside a scattering medium with the spatial resolution of ~200µm. In this project, we developed a FLOT system which was specifically customized for in-vivo brain imaging experiments.

While FLOT offers a relatively simple and non-expensive design for imaging superficial areas in the brain, still it has imaging depth limited to 2mm and its resolution drops as the imaging depth increases. To address this shortcoming, we worked on a complementary system based on the digital optical phase conjugation (DOPC) method which was shown previously that is capable of performing fluorescent tomography up to 4mm deep inside a biological tissue with lateral resolution of ~50 µm. This system also provides a non-invasive method to deliver light deep inside the brain tissue for neurostimulation applications which are not feasible using conventional techniques because of the high level of scattering in most tissue samples. In the developed DOPC system, the performance of the system in focusing light through and inside scattering mediums was quantified. We also showed how misalignments and imperfections of the optical components can immensely reduce the capability of a DOPC setup. Then, a systematic calibration algorithm was proposed and experimentally applied to our DOPC system to compensate main aberrations such as reference beam aberrations and also the backplane curvature of the spatial light modulator. In a highly scattering sample, the calibration algorithm achieved up to 8 fold increase in the PBR.

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