Using switchable fluorescent tags, MIT engineers can study how molecules in cells interact to control the cell's behavior. The researchers have developed a method that can observe up to seven different molecules at the same time, and it is even possible to observe more molecules than that.
Living cells are bombarded with a variety of molecular signals that influence the cell's behavior. Being able to measure these signals and how cells respond to them through downstream molecular signaling networks could help scientists learn more about how cells work, including what happens when cells age or become diseased.
Currently, such comprehensive studies are not possible because current cell imaging techniques are limited to imaging a few different molecular types within cells simultaneously. However, MIT researchers have developed an alternative method that can observe up to seven different molecules at once, and potentially even more than that.
Breakthroughs in Molecular Imaging Technology
"In biology, there are many examples where one event triggers a long chain of downstream events that lead to a specific cellular function," said Edward Boyden, Yihua Tan Professor of Neurotechnology. "How does this happen? This is arguably one of the fundamental questions of biology, so we wondered, could we simply watch it happen?"
The new method uses green or red fluorescent molecules that flash at different rates. By imaging cells for seconds, minutes, or hours and then using computational algorithms to extract each fluorescent signal, the amount of each target protein can be tracked over time.
Boyden, the study's senior author, is also a professor of bioengineering and of brain and cognitive sciences at MIT, a Howard Hughes Medical Institute investigator, a member of MIT's McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, and co-director of the K. Lisa Yang Center for Bionics. Qian Yong, a postdoc at MIT, is the first author of the paper.
Advances in Fluorescence Signals
Labeling molecules within cells with fluorescent proteins allows researchers to learn a lot about the functions of many cellular molecules. Such studies often use green fluorescent protein (GFP), which was first used for imaging in the 1990s. Since then, several fluorescent proteins that emit light in other colors have been developed for use in experiments.
However, typical light microscopes can only resolve two or three of these colors, and researchers can only glimpse the overall activity taking place within the cell. If more marker molecules could be tracked, researchers could measure how brain cells respond to different neurotransmitters during learning, or study the signals that drive cancer cells to metastasize.
"Ideally you could watch the signal fluctuations within the cell in real time and then understand the relationship between them. That would tell us how the cell computes," Boyden said. "The problem is, you can't look at many things at the same time."
In 2020, Boyden's lab developed a method to simultaneously image up to five different molecules within cells by targeting luminescent reporters to different locations within cells. This method, called "spatial multiplexing," allows researchers to distinguish the signals from different molecules, even if they emit the same fluorescent color.
In the new study, the researchers took a different approach: Rather than distinguishing the signals based on their physical location, they created fluorescent signals that varied over time. This technique relies on "switchable fluorophores" - fluorescent proteins that can turn on and off at specific rates. In the study, Boyden and members of his research team identified four green switchable fluorophores and then designed two others that all turn on and off at different rates. They also identified two red fluorescent proteins that switch on and off at different rates and designed an additional red fluorophore.
Each switchable fluorophore can be used to label different types of molecules within living cells, such as enzymes, signaling proteins or parts of the cytoskeleton. After imaging the cells for minutes, hours or even days, the researchers used a computational algorithm to pick out specific signals from each fluorophore, similar to how the human ear picks out different frequencies of sound.
"In a symphony orchestra, you have high-pitched instruments like flutes, and you have low-pitched instruments like tubas. In the middle are instruments like trumpets," Boyden said. "They all have different sounds, and our ears sort them out."
The mathematical technique the researchers used to analyze the fluorophore signals is called the linear nonmixing method. This method can extract different fluorophore signals, similar to how the human ear uses a mathematical model called the Fourier transform to extract different pitches in a musical piece.
Once the analysis is complete, researchers can see when and where each fluorescently tagged molecule appeared in the cell throughout the imaging process. The imaging itself can be accomplished with a simple optical microscope and no specialized equipment is required.
Explore biological phenomena
In this study, the researchers demonstrated their method by labeling six different molecules in mammalian cells that are involved in the cell division cycle. In this way, they were able to determine how levels of cyclin-dependent kinases change during the cell cycle.
The researchers also found that they could label other types of kinases involved in nearly every aspect of cell signaling, as well as cellular structures and organelles such as the cytoskeleton and mitochondria. In addition to conducting experiments using mammalian cells grown in laboratory dishes, the researchers also demonstrated that the technique works in the brains of zebrafish larvae.
The researchers say this approach is useful for observing how cells respond to any input, such as nutrients, immune system factors, hormones or neurotransmitters. It can also be used to study how cells respond to changes in gene expression or genetic mutations. All these factors play important roles in biological phenomena such as growth, aging, cancer, neurodegeneration, and memory formation.
"We can think of all of these phenomena as representing a general biological problem, where short-term events - such as ingesting a nutrient, learning something, or getting an infection - produce long-term changes," Boyden said.
In addition to conducting these types of studies, Boyden's lab is also working to expand the range of switchable fluorophores to study more signals within cells. They also hope to adapt the system so it can be used in mouse models.