New Method Enables Imaging of Signaling Networks, in Living Cells, Using Standard Microscopes

Current techniques for imaging cells are limited to investigating a handful of different molecule types within a cell at one time. But being able to better measure molecular signals, and how cells respond to them through downstream molecular signaling networks, could help scientists learn more about cellular functions. Now, researchers have developed a new method that allows them to observe up to seven different molecules at a time, at least.

This work is published in Cell in the paper, “Temporally multiplexed imaging of dynamic signaling networks in living cells.”

“There are many examples in biology where an event triggers a long downstream cascade of events, which then causes a specific cellular function,” said Edward Boyden, PhD, professor of biological engineering and of brain and cognitive sciences at MIT, a Howard Hughes Medical Institute investigator, and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research, as well as the co-director of the K. Lisa Yang Center for Bionics. “How does that occur? It’s arguably one of the fundamental problems of biology, and so we wondered, could you simply watch it happen?”

The new approach uses green or red fluorescent molecules that flicker on and off at different rates. By imaging a cell over several seconds, minutes, or hours—and extracting each of the fluorescent signals using a computational algorithm—the amount of each target protein can be tracked as it changes over time.

“Ideally, you would be able to watch the signals in a cell as they fluctuate in real time, and then you could understand how they relate to each other. That would tell you how the cell computes,” Boyden said. “The problem is that you can’t watch very many things at the same time.”

In 2020, Boyden’s lab developed a way to simultaneously image up to five different molecules within a cell, by targeting glowing reporters to distinct locations inside the cell. This spatial multiplexing approach allows researchers to distinguish signals for different molecules even though they may all be fluorescing the same color.

In the new study, the researchers created “switchable fluorophores”—fluorescent proteins that turn on and off at a specific rate. Each of these switchable fluorophores can be used to label a different type of molecule within a living cell. After imaging the cell for several minutes, hours, or even days, the researchers use a computational algorithm to pick out the specific signal from each fluorophore, analogous to how the human ear can pick out different frequencies of sound.

“In a symphony orchestra, you have high-pitched instruments, like the flute, and low-pitched instruments, like a tuba. And in the middle are instruments like the trumpet. They all have different sounds, and our ear sorts them out,” Boyden said.

The researchers then used linear unmixing to analyze the fluorophore signals. This method can extract different fluorophore signals. Once this analysis is complete, the researchers can see when and where each of the fluorescently labeled molecules were found in the cell during the entire imaging period. The imaging itself can be done with a simple light microscope, with no specialized equipment required.

The researchers demonstrated their approach by labeling six different molecules involved in the cell division cycle, in mammalian cells. This allowed them to identify patterns in how the levels of enzymes called cyclin-dependent kinases change as a cell progresses through the cell cycle.

The researchers also showed that they could label other types of kinases, which are involved in nearly every aspect of cell signaling, as well as cell structures and organelles such as the cytoskeleton and mitochondria. In addition, the researchers showed that this technique could work in the brains of zebrafish larvae.

This method could be useful for observing how cells respond to any kind of input, such as nutrients, immune system factors, hormones, or neurotransmitters, according to the researchers. It could also be used to study how cells respond to changes in gene expression or genetic mutations. All of these factors play important roles in biological phenomena such as growth, aging, cancer, neurodegeneration, and memory formation.

“You could consider all of these phenomena to represent a general class of biological problem, where some short-term event—like eating a nutrient, learning something, or getting an infection—generates a long-term change,” Boyden said.

In addition to pursuing those types of studies, Boyden’s lab is also working on expanding the repertoire of switchable fluorophores so that they can study even more signals within a cell. They also hope to adapt the system so that it could be used in mouse models.