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.
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.
The result was a simple yet versatile chip that could image C. elegans throughout all its developmental stages. To make the chips, Koushika and her postdoc Sudip Mondal poured a resin called polydimethylsiloxane into silicon wafer molds to create the device’s flexible walls. By changing the mold size and varying the amount of resin they added, they could create different-sized chambers to look at animals in various stages of growth. The resin chamber was simply glued to a coverslip and voilà: instant microfluidic imaging device.
Koushika and Mondal improved on existing devices by using water instead of gas to apply pressure to the chamber membrane. They found that gas causes bubbles to form and the light that is reflected off the bubbles decreases the resolution of the images. Water alleviates this problem.
Koushika and her team have used their microfluidic device to look at various cellular and subcellular processes in C. elegans, such as axonal transport and the migration of developing nerve cells. Because of the chamber’s ability to accommodate different-sized animals, they’ve also used it to visualize cell division in the worm’s embryos, which are as small as 0.04 mm long, and to make movies of axonal transport in a 7-mm-long fruit fly larva. Their findings were reported in the April 2011 issue of the journal Traffic. Koushika and her colleagues have also used the device to image the heartbeat of zebrafish embryos.
|A Drosophila larva crawls into the microfluidic chamber where it is immobilized, visualized, and eventually released. Video by Sudip Mondal.|
“In one talk I gave, someone in the audience referred to the device as a straitjacket,” says Koushika. She says it’s better than that. Straitjackets are more constricting, less versatile, and not as easily reversible.
Now, Koushika is working on a version of the chip that can track changes in a single animal from birth to death. She has used her current device to repeatedly immobilize and image animals over the course of an hour. The new chip would go further, allowing the animal to spend its entire life in the chamber where researchers can repeatedly immobilize it for long-term imaging and developmental studies.
She’s begun sharing her device with others. “My hope,” Koushika says, “is that as more and more people use C. elegans as a cell biological model to acquire real time data, they will turn to devices such as ours and consider them as a nice method to try for their system.”
As for her worms, she’s got them where she wants them.