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Microfluidic immobilization of physiologically active Caenorhabditis elegans

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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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

Affiliations

Authors

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.

Ethics declarations

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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