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Microfluidic immobilization of physiologically active Caenorhabditis elegans

Nature Protocols volume 5, pages 1888–1902 (2010)Cite this article

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Abstract

We present a protocol for building and operating a microfluidic device for mechanical immobilization of Caenorhabditis elegans in its physiologically active state. The system can be used for in vivo imaging of dynamic cellular processes such as cell division and migration, degeneration, aging and regeneration, as well as for laser microsurgery, Ca2+ imaging and three-dimensional microscopy. The device linearly orients C. elegans, and then completely restrains its motion by pressing a flexible membrane against the animal. This technique does not involve any potentially harmful anesthetics, gases or cooling procedures. The system can be installed on any microscope and operated using only one syringe and one external valve, making it accessible to most laboratories. The device fabrication begins by patterning photoresist structures on silicon wafers, which are then used to mold features in elastomeric layers that are thermally bonded to form the device. The system can be assembled within 3 d.

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Figure 1: Subcellular resolution imaging and optical manipulation of physiologically active C. elegans
Figure 2: Procedure overview.
Figure 3: Layout showing the masks used to fabricate the compression and flow layers of the chip.
Figure 4: Microfluidic chip for mechanical immobilization of C. elegans.
Figure 5: Fabrication of photoresist molds and PDMS layers.
Figure 6
Figure 7: Cross-section of the microfluidic device showing the immobilization region.
Figure 8: Microfluidic device integrated with off-chip components.
Figure 9: Manual operation of the microfluidic device.
Figure 10: Comparison of failed and successful fabrication of channel array.
Figure 11: Device contamination by debris and PDMS particulates.
Figure 12: Assessment of immobilization stability.
Figure 13: Lifespan analysis of the immobilized population and control population.

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References

  1. Yanik, M.F. et al. Neurosurgery: functional regeneration after laser axotomy. Nature 432, 822–822 (2004).

    Article  CAS  Google Scholar 

  2. Kerr, R., Lev-Ram, V., Baird, G., Vincent, P., Tsien, R.Y. & Schafer, W.R. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    Article  CAS  Google Scholar 

  3. Sulston, J. & Hodgkin, J. Methods. In The Nematode C. elegans (ed. Wood, W.) 587–606 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1998).

  4. Massie, M.R., Lapoczka, E.M., Boggs, K.D., Stine, K.E. & White, G.E. Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans. Cell Stress Chaperones 8, 1–7 (2003).

    Article  CAS  Google Scholar 

  5. Podbilewicz, B. & Gruenbaum, Y. Live imaging of Caenorhabditis elegans. In Live Cell Imaging: A Laboratory Manual 1st edn. Chapter 20 (eds. Goldman, R.D. & Spector, D.L.) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2005).

  6. Lewbart, G.A. & Bodri, M.S. Nematodes. In Invertebrate Medicine 1st edn. (ed. Lewbart, G.A.) (Blackwell Publishing, Ames, IA, 2006).

  7. Rohde, C.B., Zeng, F., Gonzalez-Rubio, R., Angel, M. & Yanik, M.F. Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution. Proc. Natl Acad. Sci. USA 104, 13891–13895 (2007).

    Article  CAS  Google Scholar 

  8. Zeng, F., Rohde, C.B. & Yanik, M.F. Subcellular precision on-chip small-animal immobilization, multi-photon imaging and femtosecond laser manipulation. Lab Chip 8, 653–656 (2008).

    Article  CAS  Google Scholar 

  9. Gray, J. et al. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430, 317–322 (2004).

    Article  CAS  Google Scholar 

  10. Lange, D., Storment, C., Conley, C. & Kovacs, G. A microfluidic shadow imaging system for the study of the nematode C. elegans in space. Sens. Actuators B. 107, 904–914 (2005).

    Article  CAS  Google Scholar 

  11. Heng, X. et al. Optofluidic microscopy—a method for implementing a high resolution optical microscope on a chip. Lab Chip 6, 1274–1276 (2006).

    Article  CAS  Google Scholar 

  12. Qin, J. & Wheeler, A. Maze exploration and learning in C. elegans. Lab Chip 7, 186–192 (2007).

    Article  CAS  Google Scholar 

  13. Chronis, N., Zimmer, M. & Bargmann, C. Microfluidics for in vivo imaging of neuronal and behavioral activity in C. elegans. Nat. Methods 4, 727–731 (2007).

    Article  CAS  Google Scholar 

  14. Hulme, S.E., Shevkoplyas, S.S., Apfeld, J., Fontana, W. & Whitesides, G.M. A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab Chip 7, 1515–1523 (2007).

    Article  CAS  Google Scholar 

  15. Lockery, S. et al. Artificial dirt: microfluidic substrates for nematode neurobiology and behavior. J. Neurophysiol. 99, 3136–3143 (2008).

    Article  CAS  Google Scholar 

  16. Chung, K., Crane, M.M. & Lu, H. Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nat. Methods 5, 637–643 (2008).

    Article  CAS  Google Scholar 

  17. Guo, S. et al. Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies. Nat. Methods 5, 531–533 (2008).

    Article  CAS  Google Scholar 

  18. Chokshi, T.V., Ben-Yakar, A. & Chronis, N. CO2 and compressive immobilization of C. elegans on-chip. Lab Chip 9, 151–157 (2009).

    Article  CAS  Google Scholar 

  19. Yanik, M.F. et al. Nerve Regeneration in C. elegans after femtosecond laser axotomy. IEEE J. Sel. Top. Quantum Electron. 12, 1283–1291 (2006).

    Article  CAS  Google Scholar 

  20. Steinmeyer, J. et al. Construction of a femtosecond laser microsurgery system. Nat. Protoc. 5, 395–407 (2010).

    Article  CAS  Google Scholar 

  21. Unger, M.A., Chou, H.P., Thorsen, T., Scherer, A. & Quake, S.R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113–116 (2000).

    Article  CAS  Google Scholar 

  22. Fang-Yen, C., Wasserman, S., Sengupta, P. & Samuel, A.D.T. Agarose immobilization of C. elegans. Worm Breed. Gaz. 18, 32 (2009).

    Google Scholar 

  23. Qin, D., Xia, Y. & Whitesides, G. Soft lithography and micro- and nanoscale patterning. Nat. Protoc. 5, 491–502 (2010).

    Article  CAS  Google Scholar 

  24. Lee, J.N., Park, C. & Whitesides, G.M. Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices. Anal. Chem. 75, 6544–6554 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the following funding sources: NIH Director’s New Innovator Award Program (1-DP2-OD002989), Packard Award in Science and Engineering, Sloan Award in Neuroscience, NSF Career Award, NSF Graduate Research Fellowship, NSERC Fellowship and NIH Biotechnology Training Grant. We also thank S. Quake and the Stanford Microfluidics Foundry for advice regarding device fabrication.

Author information

Author notes

  1. Cody L Gilleland and Christopher B Rohde: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Cody L Gilleland, Christopher B Rohde, Fei Zeng & Mehmet Fatih Yanik

  2. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Mehmet Fatih Yanik

Authors
  1. Cody L Gilleland
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  2. Christopher B Rohde
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  3. Fei Zeng
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  4. Mehmet Fatih Yanik
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Contributions

C.B.R. and F.Z. developed and characterized the microfluidic immobilization procedure. C.L.G. engineered the immobilization technique for manual operation, developed troubleshooting techniques and wrote the paper. C.L.G. and C.B.R. developed the other elements of the system. M.F.Y. supervised the project at all stages.

Corresponding author

Correspondence to Mehmet Fatih Yanik.

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Competing interests

The authors have filed patents on this technology. M.F.Y. is founder and chief scientific advisor of Entera Pharmaceuticals.

Supplementary information

Supplementary Figure 1

AutoCAD mask design file (ZIP 467 kb)

Supplementary Figure 2

PDF, AutoCAD mask design file, all layers (PDF 61 kb)

Supplementary Figure 3

PDF, AutoCAD mask design file, flow-1 layer (PDF 19 kb)

Supplementary Figure 4

PDF, AutoCAD mask design file, flow-2 layer (PDF 41 kb)

Supplementary Figure 5

PDF, AutoCAD mask design file, compress-1 layer (PDF 29 kb)

Supplementary Video

On-chip laser micro-surgery (MPG 436 kb)

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Gilleland, C., Rohde, C., Zeng, F. et al. Microfluidic immobilization of physiologically active Caenorhabditis elegans. Nat Protoc 5, 1888–1902 (2010). https://doi.org/10.1038/nprot.2010.143

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