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Experimental Apparatus for Nanophotonic Neuroprobe-Enabled Fluorescence Imaging

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Experimental Apparatus for Nanophotonic Neuroprobe-Enabled Fluorescence Imaging Experimental Apparatus for Nanophotonic Neuroprobe-Enabled Fluorescence Imaging by Thomas Rodrigues Lordello A thesis submitted in conformity with the requirements for the degree of Master of Applied Science The Edward S. Rogers Sr. Department of Electrical & Computer Engineering University of Toronto c Copyright 2019 by Thomas Rodrigues Lordello Abstract Experimental Apparatus for Nanophotonic Neuroprobe-Enabled Fluorescence Imaging Thomas Rodrigues Lordello Master of Applied Science The Edward S. Rogers Sr. Department of Electrical & Computer Engineering University of Toronto 2019 Fluorescence imaging has the ability to resolve brain structure and activity, in real time and with single-cell resolution. Current techniques, however, remain slow and cannot penetrate deep into the brain. This thesis investigates the use of implantable silicon photonic neuroprobes for three dimensional neuroimaging at higher framerates than what is currently possible. We present the implementation of neuroprobes as a novel method to deliver excitation in a fluorescent light sheet microscopy setup, achieving higher contrast than epi-fluorescence. We also present the algorithm and calibration of a high speed volumetric imaging setup, to monitor multiple depths simultaneously. Imaging experiments were performed on mouse brain tissues in vitro and in vivo. The system may be extended to freely moving animals in the future. ii Acknowledgements I thank my supervisor, Prof. Joyce Poon, for her unwavering support throughout the research project and this thesis, as well as for giving me the opportunity to participate in this exciting research. Without her passion and drive for seeking collaborations and pushing research forward, none of the work presented herein would have been possible. I thank our many collaborators at the Krembil Discovery Tower. Dr. Taufik Valiante for providing support for the collaboration, and to Dr. Homeira Moradi, Prajay Shah, and Dr. Anton Fomenko and others, for conducting the experiments with us. I thank Prof. Wai Tung Ng, Prof. Amr Helmy, and Prof. Willy Wong for being a part in my M.A.Sc defense committee. I thank Prof. Nazir Kherani and Prof. Stewart Aitchison for being a part of my thesis proposal committee. Finally, I thank my colleagues in the group for the fun times in the lab and outside, while being serious and helpful when needed. In particular, I would like to thank Fu Der Chen, Ilan Felts Almog, and Wesley Sacher for all their help in the neuroprobe experiments. iii Contents 1 Introduction 1 1.1 Motivation . .1 1.2 Current Imaging Techniques . .1 1.3 Silicon Photonics . .3 1.4 Thesis Objectives and Organization . .4 1.4.1 Objectives . .4 1.4.2 Organization . .4 2 Setup and Characterization 5 2.1 Neuroprobe Based Light Sheet Imaging . .5 2.1.1 Coupling Optics . .5 2.1.2 Neuroprobe . .6 2.1.3 Probe Packaging . .7 2.1.4 Imaging . .8 2.2 Synchronized 3D Imaging . .8 2.2.1 Concept . .8 2.2.2 Device Details . 10 2.2.3 Setup . 12 2.2.4 Algorithm . 12 2.2.5 Basic Implementation . 12 2.2.6 Camera Framerate . 14 2.3 Instrument Synchronization and Calibration . 15 2.3.1 Limitations . 19 3 Device Characterization 21 3.1 Waveguide Propagation Loss . 21 3.1.1 Measurement Setup . 21 3.1.2 Results . 23 3.2 Light Sheet Characterization in Fluorescein . 23 3.3 Light Sheet Characterization on Glass Slide . 24 3.3.1 Experiment . 24 iv 4 Imaging 32 4.1 Agarose Imaging . 32 4.2 in vitro Imaging . 35 4.2.1 Slice Preparation Procedure . 35 4.2.2 Imaging . 35 4.3 in vivo imaging . 38 4.3.1 Neuron Activity Detection . 39 5 Integrated Imaging 42 5.1 Background . 42 5.1.1 Concept . 42 5.1.2 Photodetectors . 42 5.1.3 Requirements . 43 5.2 Simulation . 43 5.2.1 Simulation Validation . 44 5.2.2 APD Design . 46 6 Discussion, Conclusion, and Outlook 48 6.1 Future Improvements . 48 6.1.1 3D Synchronized Imaging . 48 6.1.2 Stability . 48 6.1.3 Brain Tissue Labelling and Expression . 49 6.2 Conclusion . 49 6.3 Outlook . 49 A Solution Preparations 50 Bibliography 52 v List of Figures 1.1 Electric field magnitude for transverse electric polarized light (i.e., electric field vector along the horizontal direction. Taken from [21]). .4 2.1 Photograph of the coupling setup. The output from the laser is directed toward a MEMS mirror which selects a particular core of the fiber bundle for optical delivery. The fiber bundle is butt-coupled to the waveguides on the neuroprobe chip. Taken from [14]. .6 2.2 (a) Schematic of the neuroprobe layout. The inputs are edge couplers shown on the top right of the schematic, which are spatially addressed by selecting different cores on a fiber bundle with a MEMS mirror. These edge couplers feed into a routing network which splits the input light equally into all 4 shanks, from which it will ultimately be emitted by a set of grating couplers to form a light sheet. (b) Schematic showing the operation of the neuroprobe, where light coupled into the probe is routed to the shanks, where the grating couplers emit the light in a light sheet. Taken from [14]. .7 2.3 Photo of a packaged neuroprobe. .8 2.4 Photograph of the imaging setup. .9 2.5 Optical behaviour of the ETL. Taken from [24]. 10 2.6 (a) Diagram of the circuit used to amplify the signal from the DAQ to drive the EPC. (b) Simulated transfer characteristic of the circuit in (a) . 11 2.7 Schematic of the 3D imaging setup. 12 2.8 Plot showing linearity of the MEMS and ETL coordinates. 13 2.9 Base driving signals . 14 2.10 Driving signals for the ETL Calibration of sheet 3 at 20 Hz. (a) Whole driving signal, including reference captures and transition (b) Zoomed in to the offset optimization. Each sine period is slightly offset relative to the previous one. 17 2.11 Image SSIM as a function of timing offset introduced into the ETL signal at 20 Hz. 18 2.12 Image SSIM as a function of timing offset for: (a) MEMS x, (b) MEMS y . 18 3.1 Schematic of the test setup to measure waveguide loss. 22 3.2 Plot showing the loss increasing as length of waveguide increases. The slope of the plot is the waveguide propagation loss, in dB/mm. 23 3.3 (a) Capture of the neuroprobe in fluorescein from top, showing the light sheet in its plane of propagation. (b) Capture of the neuroprobe in fluorescein from the side, when the output is optimized for TE. (c) Capture of the neuroprobe in fluorescein from the side, when the output is optimized for TM. 25 vi 3.4 Schematic of the test setup to characterize the light sheet emitted from the probe. 26 3.5 (a) Two light sheet cross section captures overlaid as seen from the sCMOS camera. Knowing the distance traveled by the micromanipulator between the two captures, we can calculate the scale of the image in microns. (b) Center point of the light sheet as we scan the probe in the positive x direction. The slope of this plot yields the scale of the image. 27 3.6 (a) Three overlaid cross sections of the light sheet at different probe depths relative to its starting position 52 µm below the imaging plane. (b) Sample profile taken of the light sheets to show the FWHM. 28 3.7 Side view of the neuroprobe just under the glass slide coated with fluorescent film. Due to the known probe thickness, we can determine the separation between the probe and the glass slide. 29 3.8 Schematic showing the angle of emission and the difference between the FWHM measured by the camera and the FWHM of the light sheet . 30 3.9 Schematic showing the angle of emission and the difference between the FWHM measured by the camera and the FWHM of the light sheet . 31 4.1 (a) Capture of light sheet 1 using the probe as illumination. (b) Capture in same location as (a) but epi-illuminated. 33 4.2 (a) Reference capture of light sheet 3 at steady state. (b) capture of light sheet 3 while modulating the ETL and MEMS at 20 Hz, without incorporating any timing offset cal- ibration. (c) capture of light sheet 3 while modulating the ETL and MEMS at 20 Hz, after incorporating any timing offset calibration. 34 4.3 (a) Capture from a functional imaging video of a mouse brain slice, illuminated by the light sheet probe. (b) Capture in same location as (a) but epi-illuminated. 37 4.4 Time dependent fluorescence of the neurons labelled in 4.3 . ..
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