New Strategy Combines Dyes and Protein Biosensors for In Vivo Imaging

The Janelia group leaders, Eric Schreiter and Luke Lavis, at Howard Hughes Medical Institute, announced in 2021 that they had figured out a way to integrate Lavis's vivid, fluorescent Janelia Fluor dyes with Schreiter's engineered protein biosensors. Helen Farrants then redeveloped the biosensors, creating WHaloCaMP, which successfully detects calcium signals in fruit flies, zebrafish, and mice. Her work introduced a new strategy that can be applied to create sensors for tracking other physiological signals, revolutionizing biological imaging. The study was published in Nature Methods.

In theory, these sensors would allow scientists to perform imaging in live animals and track multiple physiological signals at once, two aspects of biological imaging that were challenging to accomplish with existing sensors. The scientists could track different physiological signals and brightly illuminate them in far-red light. Compared to other wavelengths, far-red light can penetrate tissues deeper and provides biologists with an extra color option beyond the standard biological imaging colors of red and green.

Everything was great, and it was fantastic, and we were happy – until we tried to use the sensors in animals, and it pretty much totally failed. It was a bit of a bummer.

Eric Schreiter, Scientist, Howard Hughes Medical Institute

Fortunately, Helen Farrants accepted the task of re-developing the protein biosensors to fulfill their original purpose, as Farrants had just arrived at Janelia for a postdoc in Schreiter's lab.

Farrants had to start from scratch to figure out a new method for the engineered protein biosensors and the JF dyes to cooperate, which allowed the team to measure physiological signals in living animals. Their initial proof-of-principle sensor, called WHaloCaMP, is able to identify calcium signals in live fruit flies, zebrafish, and mice—a crucial component of cellular communication.

The novel method can also be applied to the development of a wide range of sensors to monitor other interesting signals. The ability to observe these physiological signals in living animals may help biologists better understand how organs, tissues, and cells cooperate to perform vital tasks.

Helen started from scratch, from the ground up, and rebuilt this whole strategy for combining dyes and protein biosensors. WHaloCaMP is the first demonstration, but it won’t be the last. It is going to be a new general strategy in the field for making fluorescent biosensors to image physiology, especially in the far-red.

Eric Schreiter, Scientist, Howard Hughes Medical Institute

Forging a New Path

Finding a different method to combine the JF dye and the protein biosensor was the primary challenge that Farrants and the team had to overcome.

The group's initial sensors depended on dyes that could change form to become fluorescent. However, those dyes could not penetrate animal tissue, a problem that showed up when the researchers attempted to use the sensor in living animals but could not find any signals.

Farrants experimented with various approaches for over a year before coming up with the concept of using specific regions of the sensor protein to toggle the fluorescence on and off instead of altering the dye's composition. The bioengineered protein sensor near the attached dye was supplemented by the team with tryptophan, an amino acid.

Dyes are switched off when they come into close contact with tryptophan. The protein changes form in the presence of calcium; the tryptophan separates from the dye, and the dye becomes active.

For a year and a half, nothing worked, but I remember the day that I made this tryptophan change and I saw just the tiniest change in fluorescence when I added calcium. I knew that we at least had a starting point – we had a hook.

Helen Farrants, Postdoc, Howard Hughes Medical Institute

Seeing Signals

Tryptophan was utilized to control the fluorescence of the dye, making it possible to use dyes that are readily absorbed by living things.

The scientists demonstrated that calcium signals in live fruit flies, zebrafish, and mice could be detected using WHaloCaMP. Additionally, they demonstrated how it could be used in conjunction with other sensors to simultaneously detect up to three signals by using different colors. The scientists demonstrated the simultaneous detection of calcium signals in muscles, changes in cell glucose levels, and calcium signals in neurons in zebrafish.

To create a better version of WHaloCaMP, the team is currently collaborating with Janelia's GENIE Project Team. Together with Janelia biologists, they are utilizing the new approach to develop sensors that can identify additional physiological signals and produce sensors that contain more JF dyes. The research community at large can now access the team's biosensor development strategy, and other teams have begun to work on producing more biosensor variants.

According to Farrants, the project would not have been feasible without the communication and teamwork that take place at Janelia. Chemists like her, and others are able to create instruments that biologists both require and desire.

“I really enjoy tinkering with things and building tools, but if I know that what I am building and tinkering with has an application that someone is going to care about, I think that is what makes it fun and rewarding. That’s what I like about Janelia: You get to interact with people on a daily basis. It happens in the wider scientific world as well, but Janelia is a special place,” said Farrants

Journal Reference:

Farrants, H., et al. (2024) A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging. Nature Methods. doi.org/10.1038/s41592-024-02411-6