Optogenetics and Computer Vision for C. elegans Neuroscience and Other Biophysical Applications The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Leifer, Andrew. 2011. Optogenetics and Computer Vision for C. elegans Neuroscience and Other Biophysical Applications. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:9276708 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA © -Andrew Michael Leifer All rights reserved. Aravinthan D. T. Samuel Andrew Michael Leifer Optogenetics and computer vision for C. elegans neuroscience and other biophysical applications A is work presents optogenetics and real-time computer vision techniques to non-invasively manipulate and monitor neural activity with high spatiotempo- ral resolution in awake behaving Caenorhabditis elegans. ese methods were em- ployed to dissect the nematode’s mechanosensory and motor circuits and to elu- cidate the neural control of wave propagation during forward locomotion. Addi- tionally, similar computer vision methods were used to automatically detect and decode uorescing DNA origami nanobarcodes, a new class of uorescent reporter constructs. An optogenetic instrument capable of real-time light delivery with high spa- tiotemporal resolution to specied targets in freely moving C. elegans, the rst such instrument of its kind, was developed. e instrument was used to probe the nematode’s mechanosensory circuit, demonstrating that stimulation of a single mechanosensory neuron suffices to induce reversals. e instrument was also used to probe the motor circuit, demonstrating that inhibition of regions of cholinergic motor neurons blocks undulatory wave propagation and that muscle contractions can persist even without inputs from the motor neurons. e motor circuit was further probed using optogenetics and microuidic tech- niques. Undulatory wave propagation during forward locomotion was observed to depend on stretch-sensitive signaling mediated by cholinergic motor neurons. Specically, posterior body segments are compelled, through stretch-sensitive feedback, to bend in the same direction as anterior segments. is is the rst explicit demonstration of such feedback and serves as a foundation for under- iii Aravinthan D. T. Samuel Andrew Michael Leifer standing motor circuits in other organisms. A real-time tracking system was developed to record intracellular calcium tran- sients in single neurons while simultaneously monitoring macroscopic behavior of freely moving C. elegans. is was used to study the worm’s stereotyped reversal behavior, the omega turn. Calcium transients corresponding to temporal features of the omega turn were observed in interneurons AVA and AVB. Optics and computer vision techniques similar to those developed for the C. elegans experiments were also used to detect DNA origami nanorod barcodes. An optimal Bayesian multiple hypothesis test was deployed to unambiguously classify each barcode as a member of one of distinct barcode species. Overall, this set of experiments demonstrates the powerful role that optoge- netics and computer vision can play in behavioral neuroscience and quantitative biophysics. iv Contents Introduction . Background .............................. .. C. elegans as a model organism ............... .. Optical neurophysiology ................... .. Real-Time Computer Vision ................. .. Applications to Behavioral Neuroscience .......... .. Additional applications of computer vision for molecular biophysics .......................... .. Onwards ........................... Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans . Introduction ............................. . Results ................................ .. Experimental Setup ..................... .. Spatial resolution of the illumination system ........ .. Optogenetic manipulation of muscle cells ......... .. Optogenetic manipulation of cholinergic motor neurons . .. Optogenetic manipulation of single touch receptor types . . Discussion .............................. . Methods ............................... .. Strains ............................ .. Microscopy .......................... .. Optics and illumination ................... .. Software ........................... v .. Behavioral experiments ................... .. Quantifying locomotory behavior .............. . Acknowledgements .......................... . Accompanying Videos ........................ .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. .. Video ............................. . Design Considerations ........................ .. Guiding Principles ...................... .. Choosing a DMD ....................... .. Schemes for segmentation and targeting .......... .. Computer vision implementation .............. .. Camera selection ....................... .. Data Storage and organization ............... . Manuscript Information ....................... .. Previously Published As ................... .. e Author’s Contribution .................. Bending waves during Caenorhabditis elegans locomotion are driven and organized by proprioceptive coupling . Introduction ............................. . Results ................................ .. e bending of one body region requires the bending of its anterior neighbor ...................... .. Muscle activity is positively correlated with the curvature of adjacent anterior neighbors ................ .. Post-channel body curvature follows channel curvature with a viscosity- dependent delay .............. vi .. Stretch-sensitive feedback requires cholinergic motor neu- rons ............................. .. Body wall muscles exhibit hysteresis ............ . Discussion .............................. . Material and Methods ........................ .. Worm strains ......................... .. Microuidic devices ..................... .. Measuring locomotion of partially immobilized worms .. .. Ivermectin-induced paralysis. ................ .. Calcium imaging of body wall muscle activities. ...... .. Optogenetic stimulation. .................. . Accompanying Video ......................... .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ............................ .. Video ........................... .. Video ........................... .. Video ........................... . Supplementary Materials ...................... .. Parallels to songbird motor pathway ............ . Manuscript Information ....................... .. Submitted for publication as ................. .. e Author’s Contribution .................. Sub-micrometer Geometrically Encoded Fluorescent Barcodes Self- Assembled from DNA . Introduction ............................. . A Simple DNA Origami Barcode ................... vii .. Construction of Simple Barcode ............... .. Inspecting the simple barcodes ............... .. Quantication of the yield of the simple barcodes ..... . Dual-labeled DNA origami Barcodes ................. .. Inspecting the dual-labeled barcodes ............ . DNA origami barcode system requiring super-resolution microscopy .. Introduction to super-resolution microscopy techniques . .. DNA-PAINT-based DNA origami barcodes ......... . More Complex Geometries ...................... .. DNA-PAINT-based DNA origami barcodes ......... . DNA Barcodes attached to cells ................... . Discussion .............................. . Future Directions ........................... . Materials and Methods ........................ .. Materials ........................... .. Design of the nano-barcodes ................ .. Self-assembly of the nano-barcodes ............. .. Tagging yeast cells with nano-barcodes ........... .. Sample preparation for uorescence imaging ........ .. Diffraction-limited uorescence imaging .......... .. DNA-PAINT Imaging .................... .. Automated characterization of the geometry of BRG and GRG barcodes ........................ .. Decoding software for diffraction-limited TIRF images .. . Acknowledgements .......................... . Manuscript Information ....................... .. Submitted for Publication As ................ .. Author Contributions .................... . Supplementary Material ....................... Automated DNA origami barcode detection using Bayseian multi- ple hypothesis testing . Introduction ............................. . System and Methods ......................... viii .. DNA origami nanorod barcodes ............... .. Sample Preparation ..................... .. Imaging ............................ .. Task at Hand ......................... . eory ................................. .. Conditioning