We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (∼50 frames per second) to provide high spatial resolution (∼30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.
Bibliographical noteFunding Information:
This work was supported by the Dana Foundation, US National Science Foundation and a US National Insitutes of Health Pioneer Award to A.D.T.S. A.M.L. is supported by a National Science Foundation Graduate Research fellowship. We thank M. Zhen (Samuel Lunenfeld Institute), N. Ringstad (Skirball Institute of Biomolecular Medicine, New York University School of Medicine), A. Gottschalk (Frankfurt Molecular Life Sciences Institute) and B. Neumann and M. Hilliard (Queensland Brain Institute, University of Queens) for gifts of transgenic strains; J. Stirman for sharing unpublished results about a similar system that he developed; B. Chow and T. Lindsay for useful discussions; and A. Tang and B. Schwartz for assistance with data analysis.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology