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MIT observes multiple fluorescence signals from the same living cell

29 Nov 2023

Temporal multiplex method tracks different emitters to image cellular responses.

Living cells receive and transmit molecular signals in complex patterns, and imaging those patterns effectively using fluorescence techniques can be challenging.

Imaging many signals at once in the same living cell would be a useful strategy, but although multiplexed fluorescence imaging has developed to the point where multiple emissions can be recorded, the equipment involved has been expensive and demanding.

A project at MIT has now demonstrated a way to use fluorophores with different properties to indicate different cellular signals using conventional hardware, and published the findings in the journal Cell.

"There are many examples in biology where an event triggers a long downstream cascade of events, which then causes a specific cellular function," said MIT's Edward Boyden. "How does that occur? It’s arguably one of the fundamental problems of biology. So we wondered, could you simply watch it happen?"

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

Boyden's new study employs a temporal multiplexing approach, making use of fluorescent molecules that switch on and off at different rates and emit different colors, according to MIT. By imaging a cell over several timescales from seconds to hours, and then extracting each of the fluorescent signals using a computational algorithm, the amount of each target protein can be tracked as it changes over time.

Details of cell growth, aging and disease

For this study, the project identified four green switchable fluorophores and then engineered two more, all of which turn on and off at different rates. They also identified two red fluorescent proteins that switch at different rates and engineered one additional red fluorophore. Each of these can be used to label a different type of molecule within a living cell, such as an enzyme, signalling protein, or part of the cell cytoskeleton.

After imaging over different periods of time, an operation the project termed temporally multiplexed imaging (TMI), the researchers used linear unmixing computational analysis to pick out the specific signal from each fluorophore, in a manner analogous to the way that human ears can pick out different frequencies of sound.

With this analysis complete, researchers could 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.

In trials, MIT demonstrated its approach by labeling six different molecules involved in the cell division cycle in mammalian cells, and identified patterns in how the levels of particular enzymes, called cyclin-dependent kinases, changed as a cell progresses through the cycle.

Boyden, leader of the MIT Synthetic Neurobiology Group and one of the original pioneers of optogenetics as a way to study neural activity, anticipates the TMI technique delivering valuable data about how cells respond to nutrients, immune system factors or neurotransmitters. It could also help to show details of growth, aging, cancer or neurodegeneration.

"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," noted Boyden.

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