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  • Don’t move: Improving immobilization for 3-D imaging and laser surgery

Jun 2008
Gary Boas

Researchers often turn to the small semitransparent organism known as C. elegans for investigations of disease models, including, for example, Parkinson’s and Alzheimer’s diseases and muscular dystrophy. However, the traditional approaches to using C. elegans as a model system have proved ill-suited to high-throughput investigations.

In a PNAS paper published last year, Mehmet Fatih Yanik, a researcher at MIT in Cambridge, Mass., and colleagues reported using microfluidic systems to demonstrate high-throughput on-chip small-animal screening for whole-animal studies of diseases such as neural degeneration. Following publication, they received e-mails from researchers asking about another longstanding challenge: complete immobilization of the animals. This is typically achieved using anesthesia, which can have detrimental or uncharacterized side effects on biological processes and is not well-suited to high-throughput screening because it requires a significant amount of time.


Researchers have described a microfluidic device that facilitates complete immobilization of C. elegans, a model organism for whole-animal studies of disease. It achieves this through a combination of aspiration channels and a sealing membrane. Without suction, the worm is free to move in the microfluidic channel (a). Fluidic pressure in the “press down” channel is increased, which flexes the membranes that separate the channels downward, sealing and immobilizing the animal (b). Reprinted with permission of Lab on a Chip.

In Vol. 8, issue 5, of Lab on a Chip, they report an improvement to the technique that facilitates immobilization of animals for several minutes at a time — as opposed to just a few seconds, as was the case in their earlier study. The authors use this method to perform three-dimensional imaging of cells in awake animals as well as “100 percent” repeatable subcellular precision laser micro- and nanosurgery.

The microfluidic device contains a 100-μm-deep flow channel with a number of 15-μm-deep aspiration channels. The animals are captured and aligned by lowering the pressure in the aspiration channels. Previous studies had used such aspiration channels, but these alone afford only partial immobilization. To achieve full immobilization, Yanik and colleagues created a seal around the animals with a 15- to 25-μm-thick flexible sealing membrane.

The researchers performed several experiments to show the degree of immobilization using the device. They performed femtosecond laser microsurgery with a modified Nikon microscope and two-photon imaging with a Spectra-Physics femtosecond laser; they used a Photometrics camera for wide-field imaging. First, they acquired images of an animal’s anterior ventral mechanosensory cell body and its axon at three time points, 5 s apart. Even at 503 magnification, the images revealed very little movement. Then, to determine the utility of the technique versus anesthesia specifically, they labeled the cell bodies of touch neurons with GFP and tracked them using a software algorithm. Here again, they observed very little movement, comparable even with the degree of immobilization achieved with deeply anesthetized animals.

After immobilizing the worms (left), the researchers performed two-photon imaging of them (middle) as well as femtosecond microsurgery (right).

A number of applications could benefit from use of the device, as could other technologies the researchers have developed. Cellular-resolution imaging is needed for almost all biological assays using C. elegans, Yanik explained; in particular, the technologies will allow investigators to perform in vivo neural regeneration studies on awake animals both rapidly and precisely.

“As a bonus,” he said, “the technologies can dramatically accelerate all large-scale genetic/drug studies on most disease models. We are currently using large-scale genetic/drug libraries to discover factors that affect neural regeneration in vivo.”

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