An official website of the United States government
Controllable, precise, and stable rotational manipulation of model organisms is valuable in many biomedical, bioengineering, and biophysics applications. We present an acoustofluidic chip capable of rotating Caenorhabditis elegans ( C. elegans ) in both static and continuous flow in a controllable manner. Rotational manipulation was achieved by exposing C. elegans to a surface acoustic wave (SAW) field that generated a vortex distribution inside a microchannel. By selectively activating interdigital transducers, we achieved bidirectional rotation of C. elegans , namely counterclockwise and clockwise, with on-demand switching of rotation direction in a single chip. In addition to continuous rotation, we also rotated C. elegans in a step-wise fashion with a step angle as small as 4° by pulsing the signal duration of SAW from a continuous signal to a pulsed signal down to 1.5 ms. Using this device, we have clearly imaged the dopaminergic neurons of C. elegans with pdat-1:GFP expression, as well as the vulval muscles and muscle fibers of the worm with myo-3::GFP fusion protein expression in different orientations and three dimensions. These achievements are difficult to realize through conventional ( i.e. , non-confocal) microscopy. The SAW manipulations did not detectably affect the health of the model organisms. With its precision, controllability, and simplicity in fabrication and operation, our acoustofluidic devices will be well-suited for model organism studies.
more »
« less
Fluorescence-based sorting of Caenorhabditis elegans via acoustofluidics
Effectively isolating and categorizing large quantities of Caenorhabditis elegans ( C. elegans ) based on different phenotypes is important for most worm research, especially genetics. Here we present an integrated acoustofluidic chip capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separate them accordingly in a high-throughput manner. Utilizing planar fiber optics as the detection unit, our acoustofluidic device requires no temporary immobilization of worms for interrogation/detection, thereby improving the throughput. Implementing surface acoustic waves (SAW) as the sorting unit, our device provides a contact-free method to move worms of interest to the desired outlet, thus ensuring the biocompatibility for our chip. Our device can sort worms of different developmental stages (L3 and L4 stage worms) at high throughput and accuracy. For example, L3 worms can be processed at a throughput of around 70 worms per min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms per min. In our acoustofluidic chip, the time period to complete the detection and sorting of one worm is only 50 ms, which outperforms nearly all existing microfluidics-based worm sorting devices and may be further reduced to achieve higher throughput.
more »
« less
- Award ID(s):
- 1807601
- PAR ID:
- 10172988
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 20
- Issue:
- 10
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 1729 to 1739
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
We developed an innovative paper-based platform for high-throughput culturing, trapping, and monitoring of C. elegans. A 96-well array was readily fabricated by placing a nutrient-replenished paper substrate on a micromachined 96-well plastic frame, providing high-throughput 3D culturing environments and in situ analysis of the worms. The paper allows C. elegans to pass through the porous and aquatic paper matrix until the worms grow and reach the next developmental stages with the increased body size comparable to the paper pores. When the diameter of C. elegans becomes larger than the pore size of the paper substrate, the worms are trapped and immobilized for further high-throughput imaging and analysis. This work will offer a simple yet powerful technique for high-throughput sorting and monitoring of C. elegans at a different larval stage by controlling and choosing different pore sizes of paper. Furthermore, we developed another type of 3D culturing system by using paper-like transparent polycarbonate substrates for higher resolution imaging. The device used the multi-laminate structure of the polycarbonate layers as a scaffold to mimic the worm’s 3D natural habitats. Since the substrate is thin, mechanically strong, and largely porous, the layered structure allowed C. elegans to move and behave freely in 3D and promoted the efficient growth of both C. elegans and their primary food, E. coli. The transparency of the structure facilitated visualization of the worms under a microscope. Development, fertility, and dynamic behavior of C. elegans in the 3D culture platform outperformed those of the standard 2D cultivation technique.more » « less
-
Small roundworms such as Caenorhabditis elegans release chemical signals called ascarosides in order to communicate with other worms of the same species. Using the ascarosides, the worm can tell its friends, for example, how crowded the neighborhood is and whether there is enough food. The ascarosides thus help the worms in the population decide whether the neighborhood is good – meaning they should hang around, eat, and make babies – or whether the neighborhood is bad. If so, the worms should develop into a larval stage specialized for dispersal that will allow them to find a better neighborhood. Roundworms make the ascarosides by attaching a long chemical ‘side chain’ to an ascarylose sugar. Further chemical modifications allow the worms to produce different signals. In general, to signal a good neighborhood, worms attach a structure called an indole group to the ascarosides. To signal a bad neighborhood, worms make the side chain very short. But how does a worm control which ascarosides it makes? Zhou, Wang et al. now show that C. elegans can change the meaning of its chemical message by modifying the ascarosides that it has already produced instead of making new ones from scratch. Specifically, as their neighborhood runs out of food, C. elegans can use an enzyme called ACS-7 to initiate the shortening of the side chains of indole-ascarosides. The worm can thus change a favorable ascaroside signal that causes the worms to group together into an unfavorable ascaroside signal that causes the worms to enter their dispersal stage. Although Zhou, Wang et al. have focused on chemical communication in C. elegans, the findings could easily apply to the many other species of roundworm that produce ascarosides. Knowing how worms communicate will help us to understand how worms respond to their environment. This knowledge could potentially be used to interfere with the lifecycles and survival of parasitic worm species that harm health and crops.more » « less
-
Abstract Mechanical properties have emerged as a significant label-free marker for characterizing deformable particles such as cells. Here, we demonstrated the first single-particle-resolved, cytometry-like deformability-activated sorting in the continuous flow on a microfluidic chip. Compared with existing deformability-based sorting techniques, the microfluidic device presented in this work measures the deformability and immediately sorts the particles one-by-one in real time. It integrates the transit-time-based deformability measurement and active hydrodynamic sorting onto a single chip. We identified the critical factors that affect the sorting dynamics by modeling and experimental approaches. We found that the device throughput is determined by the summation of the sensing, buffering, and sorting time. A total time of ~100 ms is used for analyzing and sorting a single particle, leading to a throughput of 600 particles/min. We synthesized poly(ethylene glycol) diacrylate (PEGDA) hydrogel beads as the deformability model for device validation and performance evaluation. A deformability-activated sorting purity of 88% and an average efficiency of 73% were achieved. We anticipate that the ability to actively measure and sort individual particles one-by-one in a continuous flow would find applications in cell-mechanotyping studies such as correlational studies of the cell mechanical phenotype and molecular mechanism.more » « less
-
A worm called Caenorhabditis elegans has a nervous system made up of only 302 neurons, far fewer than the billions of cells that comprise our own brains. And yet these few hundred neurons are enough for these worms to detect and respond to their surroundings. C. elegans is thus a popular choice for studying how nervous systems process sensory information and use it to control behavior. Yet, most experiments to date have used only simple stimuli, such as taps or pokes, and studied a handful of behaviors, such as whether or not a worm stops moving or backs up. This limits the conclusions it has been possible to draw. Liu et al. therefore set out to determine how the worm’s nervous system responds to more complex stimuli. These included physical stimuli, such as taps on the side of the dish containing the worms, as well as simulated stimuli. To generate the latter, Liu et al. used a technique called optogenetics to directly activate the neurons in the worm’s body that would normally detect information from the senses, by simply shining a light on the worms. Doing so gives the worm the sensation of a physical stimulus, even though none was present. Liu et al. then used mathematics to examine the relationships between the stimuli and the worms’ responses. The results confirmed that worms usually respond to simple stimuli, such as taps on the side of their dish, by backing up. But they also revealed more advanced forms of stimulus processing. The worms responded differently to stimuli that increased over time versus decreased, for example. A worm's response to a stimulus also varied depending on what the worm was doing at the time. Worms that were in the middle of turns, for instance, ignored stimuli to which they would normally respond. This suggests that an animal’s current behavior influences how its nervous system interprets sensory information. The discovery of relatively sophisticated responses to sensory stimuli in C. elegans indicates that even simple nervous systems are capable of flexible sensory processing. This lays a foundation for understanding how neural circuits interpret sensory signals. Building on this work will ultimately help us understand how more complicated nervous systems interpret and respond to the world.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d0lc00051e
