A mini-review on C. elegans adult worms immobilization using microfluidics

Immobilization of C. elegans during live cell imaging: rational

With its unique transparency features and detailed biology, C. elegans represents a model of choice for biologists to study a whole animal using live cell imaging. C. elegans immobilization is a pre-requisite to produce high-resolution live-cell video and/or images. The aim is clearly to prevent, as much as possible, movement-generated artifacts: e.g worm being out-of focus or moving outside the recorded field.

Several non-microfluidics chemical or physical immobilization techniques have been reported and described (for an extensive review see Aufderheide, 2008) in the literature: anesthetics drugs, cold, glue and more recently polysterene nanoparticles (Kim et al., 2013).

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Although they have proved valuables, all of these procedures show a number of limitations. The use of chemical compounds, induces physiological modifications, which may interfere with the biological question studied. Also, with these techniques, C. elegans worms do not have identical orientation, they may not be easily accessible for further manipulations. While the use of polysterene nanoparticles is easy to set-up, one caveat is that immobilization is dependent on worm growth stages (Kim et al., 2013).

Microfluidic device active methods

A/ The compressive method

This technique takes advantage of the physical properties of PDMS membrane. In this setting, the microfluidic device contains integrated valves. The C. elegans worm is placed in the bottom flow layer channel, with applied-pressure the membrane is deflected and as a result the worm is physically immobilized. A pressure adjustment allows the use of different worm developmental stages. Pressure applied during immobilization may not be compatible with a long immobilization period. Furthermore, the caveats with this technique is that it is not user friendly, it is tricky to generate and the set-up unless one establishes a collaboration with a well trained microfluidic research group. For biologists users, we recommend the use of the passive method, which is more easy to handle.

B/ The microfluidic temperature-induced immobilization

This method described in Chung et al., 2008 allows a strict immobilization of C. elegans, stopping all internal movement and thus allowing sub-cellular structures imaging. In this setting (see figure) the worm is placed in the bottom flow channel (red), two micro valves control the flow and the worm placement (green), and a temperature-controlled liquid flow channel is placed on top of the worm channel allowing a rapid cool-down of C.elegans temperature. While this method is very useful, it implies a total immobilization of the worm and hence it is not adequate for live-animal video imaging. However, with appropriate devices temperature cool down can be reversed. Also, the use of a worm sorter may prove necessary to get the worms in the same orientation.

C. elegans Immobilization using microfluidic devices: a time-saving strategy!

The main reason why experimentator will want to use microfluidics is for time-saving considerations and reproducible immobilization conditions. Microfluidics allows a fast handling and consistent immobilization of C. elegans. (San-Miguel A and Lu H, 2013). Moreover, microfluidic immobilization presents several advantages among which unidirectional orientation, no superposition of worms, further manipulation can be performed: such as drug injection or ultra fast temperature control of C. elegans worm environment.

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Microfluidic passive method : the worm trap

Conclusion: C.elegans immobilization

Using microfluidic devices for C. elegans immobilization is time saving: with rapid handling, immobilization and imaging time spent per worm experimentation. It allows you to eliminate the use of anesthetics, which perturbed animal physiology. Depending on your needs, you may want to choose an easy to set up method like microfluidic worm traps, or if you need to have a complete block of both external and internal worm movement you’ll find that this cooling device may be more suitable.

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References

Aufderheide, KJ. (2008). An overview of techniques for immobilizing and viewing living cells. Micron 39, 71–76

Kim E, Sun L, Gabel CV, Fang-Yeng C. (2013). Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization. PLoS ONE 8(1): e53419

San-Miguel A and Lu H , Microfluidics as a tool for C. elegans research*(September 24, 2013), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.7.1.

Hulme, S.E., Shevkoplyas, S.S., Apfeld, J., Fontana, W., and Whitesides, G.M. (2007). A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab Chip7, 1515-1523.

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

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

S. Ben-Aroya, X. Pan, JD. Boeke, and P. Hieter, Making temperature-sensitive mutants, Methods Enzymol. 2010