Immobilizing Caenorhabditis elegans for high-resolution imaging.

A worm swims into the holding chamber, which is mounted on a glass coverslip. With gentle pressure applied to the top of the device, the walls bow inward, squeezing and immobilizing the animal. The chip is then placed in a microscope and the animal is imaged. After the data are collected, the pressure is released and the animal swims away unharmed.

They are active little creatures. Look at Caenorhabditis elegans through a microscope and you’ll see the worms slither to and fro. Getting them to hold still is like trying to herd cats. But HHMI international early career scientist Sandhya P. Koushika devised an inexpensive, simple way to get the worms to pause so she can image cellular activity in the transparent creatures.

In neurons, many proteins and organelles need to be transported from the cell body where they are made down the length of the axon to the synapse, the junction between two nerve cells. This process involves an array of helper proteins that do everything from regulating transport to carrying cargo. Koushika and her team at the National Center for Biological Sciences in Bangalore, India, study the roles of these helper proteins by genetically altering them in C. elegans and then watching what happens under a microscope.

Koushika uses the worms to study neuronal transport because they are small—about 1 mm long—transparent, and easy to manipulate genetically. Their neuronal circuitry is also very well defined. But she needed a way to get them to stay still long enough to capture a snapshot of the activity inside their neurons.

She tried anesthetizing the worms, to no avail. “Nothing was moving—neither the worm nor the cargo in the axons,” explains Koushika. Gluing the worms to a coverslip—which was nearly impossible with the miniscule animals and tinier embryos—turned out to be a bad idea. The worms were immobilized, but the glue was toxic.

Koushika needed to create a new microimaging system. Her solution: modify a miniature microfluidic chip that other groups had developed to look at behavior and cellular processes in C. elegans.

The imaging chips consisted of a small holding chamber mounted on a glass coverslip. Once the animal swims into the chamber, gentle pressure is applied to the top of the device. Because the chamber is made of flexible material, the pressure causes the walls to bow inward, squeezing and immobilizing the animal—just enough to hold it still without squishing it. The chip is then placed in a microscope and the animal is imaged. After the data are collected, the pressure is released and the animal swims away unharmed.

A C. elegans worm is seen inside Sandhya P. Koushika’s microfluidic chip prior to trapping.
Image: Jyoti Dubey

Because most microfluidic chips had been designed to look at large, stationary structures like nerves, Koushika needed to create something with higher resolution that could hold her worms still long enough to track molecules in motion within the tiniest embryos.

And her lab was not a microfluidics lab, so the complexity of the system had to be low. “We had to develop something that was easy to make and easy to use that worked reliably every single time,” she says. Koushika also needed the device to visualize subcellular processes at high resolution, but it couldn’t require a lot of technical support or high-end facilities.

Image: VSA Partners

	PAGE 1 of 2	Continue