New biosensor illuminates physiological signals in living animals

Eric Schreiter and Luke Lavis thought they had found the solution. In 2021, Janelia Group executives announced they had developed a way to combine the protein biosensors designed by Schreiter with Lavis’s Janelia Fluor fluorescent dyes.

These sensors, which can detect different physiological signals and illuminate them with far-red light, would theoretically allow scientists to image living animals and track multiple physiological signals simultaneously, two aspects of biological imaging that are difficult to achieve with existing sensors. Far-red light can penetrate deeper into tissue than other wavelengths, and it gives scientists an additional color to use beyond the typical hues, such as green and red, used in biological imaging.

“Everything was going great, it was fantastic, and we were happy, until we tried to use the sensors on animals, and it failed,” Schreiter recalls. “It was a bit disappointing.”

Fortunately, Helen Farrants had just arrived at Janelia for her postdoctoral fellowship in Schreiter’s lab, and she accepted the challenge of redeveloping the protein biosensors to achieve their original intent.

Starting from scratch, Farrants created a new way to make protein biosensors and JF dyes work together, which allowed the team to achieve their goal of measuring physiological signals in living animals. Their first proof-of-principle sensor, dubbed WHaloCaMP, can detect calcium signals—a key component of cellular communication—in living fruit flies, zebrafish, and mice.

This new technique can also be used to create a range of sensors to track other signals of interest. The ability to observe these physiological signals in living animals could give biologists insight into how cells, tissues and organs work together to perform important functions.

“Helen started from scratch, from scratch, and rebuilt this whole strategy to combine dyes and protein biosensors,” Schreiter says. “WHaloCaMP is the first demonstration, but it won’t be the last. It really will be a new general strategy in the field of making fluorescent biosensors for physiological imaging, particularly in the far red.”

Opening a new path

The main hurdle Farrants and his team had to overcome was finding another way to make the protein biosensor and the JF dye work together.

The first sensors the team created used dyes that fluoresced. However, these dyes couldn’t penetrate animal tissue, a problem that became apparent when the team tried using the sensor on live animals and failed to detect any signals.

After experimenting with different strategies for more than a year, Farrants came up with the idea of ​​using specific parts of the sensor protein to turn the fluorescence on or off, rather than using a shape-shifting dye. The team added an amino acid called tryptophan to the bioengineered protein sensor near the attached dye. When the dye came into close contact with the tryptophan, the dye turned off. In the presence of calcium, the protein changed shape: the tryptophan moved away from the dye, and the dye turned on.

“For a year and a half, nothing worked, but I remember the day I made that change to tryptophan and saw a tiny change in fluorescence when I added calcium. I knew we had at least a starting point, we had a hook,” Farrants recalls.

See the signals

The use of tryptophan to modulate dye fluorescence allowed the use of dyes that were readily absorbed by tissues and used in living animals.

The researchers showed that the WHaloCaMP could be used to detect calcium signals in live fruit flies, zebrafish, and mice. They also showed that it could be used with other sensors to detect up to three signals at once using distinct colors. In zebrafish, they showed that they could simultaneously detect glucose changes in cells, calcium signals in muscles, and calcium signals in neurons.

The team is currently working with Janelia’s GENIE project team to develop an improved version of WHaloCaMP. They are also working with biologists at Janelia to use the new strategy to develop sensors to detect other physiological signals and to create sensors with additional JF dyes. The team has also made its biosensor construction strategy available to the broader scientific community, and other groups have begun developing additional versions of the sensors.

Farrants says the project would not have been possible without the interaction and collaboration that occurs at Janelia, which allows him and other chemists to create tools that biologists need and want.

“I really enjoy tinkering and building tools, but if I know that what I’m building and tinkering with has an application that someone is going to be interested in, I think that’s what makes it fun and rewarding,” Farrants says. “That’s what I love about Janelia: you get to interact with people on a daily basis. That happens in the broader scientific world as well, but Janelia is a special place.”