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. 2013;8(1):e53419.
doi: 10.1371/journal.pone.0053419. Epub 2013 Jan 3.

Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization

Eric Kim  1 , Lin Sun, Christopher V Gabel, Christopher Fang-Yen
Affiliations

Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization

Eric Kim et al. PLoS One. 2013.

Abstract

One advantage of the nematode Caenorhabditis elegans as a model organism is its suitability for in vivo optical microscopy. Imaging C. elegans often requires animals to be immobilized to avoid movement-related artifacts. Immobilization has been performed by application of anesthetics or by introducing physical constraints using glue or specialized microfluidic devices. Here we present a method for immobilizing C. elegans using polystyrene nanoparticles and agarose pads. Our technique is technically simple, does not expose the worm to toxic substances, and allows recovery of animals. We evaluate the method and show that the polystyrene beads increase friction between the worm and agarose pad. We use our method to quantify calcium transients and long-term regrowth in single neurons following axotomy by a femtosecond laser.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nanoparticle-mediated immobilization.
(a) Transfer molten agarose to slide containing plastic spacers (shown in gray). (b) Quickly add second slide to form pad. (c) After agarose has cooled, remove upper slide and spacers. (d) Add polystyrene nanoparticles. (e) Add washed worm(s). (f) Add coverslip. The worms are now ready for imaging. (g) Seal coverslip with wax if necessary. (h) To recover animals, lift coverslip upwards without sliding.
Figure 2
Figure 2. Quantifying movement during immobilization.
(a) Fluorescence of AFD soma in a nanoparticle-immobilized Pgcy-8::YC3.60 worm. Centroid of cell body indicated by crosshairs. Scale bar: 20 µm. (b) NP: Mean absolute displacement of AFD neuron cell body during 10 s intervals in animals immobilized with 0.1 µm diameter nanoparticles, as a function of agarose concentration in pad. NGM: no beads. (c) Mean absolute displacement of buccal cavity during 10 s intervals for adult worms on 10% agarose pads and varied bead diameters; L1, L3, and adult (Ad) worms immobilized with 0.1 µm beads on 10% agarose pads.
Figure 3
Figure 3. Compression during immobilization.
(a) Adult hermaphrodite in NGM buffer. (b) Same animal during immobilization on 10% agarose pad. Arrows indicate measurement of dorso-ventral width. Scale bars: 100 µm. (c) Dorso-ventral width as a function of agarose concentration for 0.1 µm diameter nanoparticle-immobilized worms (n>5 for each point).
Figure 4
Figure 4. Nanoparticles reduce locomotory rate in a surface-dependent manner.
Locomotory frequency of freely swimming worms and worms in contact with 0.5% agarose pads, in the presence (NP) and absence (NGM) of nanoparticles. n = 30 for each group. **p<0.001.
Figure 5
Figure 5. In vivo laser axotomy and time-lapse imaging.
a) Images from a continuously immobilized C. elegans, showing an ALM neuron severed 20 µm from the cell soma (top panel) immediately following laser axotomy and (bottom panel) after 10 hr. Arrow indicates lesion point. b) Average regenerative outgrowth of ALM neurons severed at the indicated distances. Outgrowth was measured from images taken at 10 hr post surgery and categorized as initiating from the lesion point or from the cell soma (* indicates p<0.05 by Student’s t-test). c) Average FRET signals measuring cellular calcium response to laser axotomy. At each time point, two channel fluorescent FRET images of the target neuron where averaged across the cell soma and along the axon within 5 µm of the lesion point to measure intracellular calcium levels in those regions. Arrows indicate time of laser damage (t = 0 s). Shaded regions indicate standard error on the mean at each time point. n indicates number of worms assayed.
See this image and copyright information in PMC

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