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A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

Abstract

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.

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Figure 1: Optical configuration of the system and components.
Figure 2: Custom software for the real-time illumination of freely behaving C. elegans.
Figure 3: Modifications of the 3-LCD projector to limit the spectral width of the RGB colors.
Figure 4: Disassembly and insertion of custom optics into the 3-LCD projector.
Figure 5: Characterization of the completed illumination system.
Figure 6: Example application: selected area illumination of C. elegans.

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Acknowledgements

We thank members of the Caenorhabditis Genetic Center, W. Schafer, Y. Tanizawa (Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK) and E. Boyden (Massachusetts Institute of Technology) for reagents; the US National Institutes of Health (H.L.), the Alfred P. Sloan Foundation (H.L.), the Human Frontier Science Program Organization (S.J.H.), the Deutsche Forschungsgemeinschaft (grants GO1011/2-1, SFB807-P11, FOR1279-P1, EXC115/1) and the Schram Foundation (A.G.) for funding. We also thank J. Andrews and B. Parker in the Georgia Institute of Technology School of Chemical and Biomolecular Engineering machine shop and D. Woodyard in the glass shop.

Author information

Authors and Affiliations

Authors

Contributions

J.N.S. designed and constructed the illumination system. J.N.S. and M.M.C. characterized the system. S.J.H. and A.G. contributed to reagents and provided valuable discussions. J.N.S., M.M.C., S.J.H., A.G. and H.L. designed the experiments. J.N.S. and H.L. prepared the manuscript.

Corresponding author

Correspondence to Hang Lu.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Measurement of temporal resolution and accuracy. (DOCX 115 kb)

Supplementary Table 1

Description and measurements for filters used for internal insertion into Hitachi CP-X605. (DOCX 138 kb)

Supplementary Note 1

Measuring temporal resolution, temporal and spatial accuracy. (DOCX 13 kb)

Supplementary Note 2

Correct sizing of custom filters. (DOCX 11 kb)

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Stirman, J., Crane, M., Husson, S. et al. A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents. Nat Protoc 7, 207–220 (2012). https://doi.org/10.1038/nprot.2011.433

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