AsianScientist (Jul. o1, 2023) –In recent years, scientists have developed sophisticated tools to look inside living cells in ever-finer detail. However, these images are generally frozen in time and, thus, capture only a slice of the dynamic and messy reality of what’s happening in the cells. In a study published in the Journal of Cell Biology, Kyoto University researchers report a new method to visualize cellular dynamics down to the level of single molecules.

Biologists use single fluorescent-molecule imaging (SFMI) techniques to study cellular processes at the resolution of single molecules by tagging them with light-emitting probes. The new method captures images of single cells at up to 10,000 frames per second, enabling unprecedented time resolution for SFMI. This provides biologists with a way to study how supramolecular structures within cells, say membrane domains or functional membrane organelles, are organized at the level of single molecules.

“We can figure out how constant molecules enter and go out from [organellar] structures, which is very important to understand how such structures are organized or controlled in response to the stimuli or extracellular signals,” said Takahiro Fujiwara, a biophysicist and lead author of the paper in an interview Asian Scientist Magazine.

The researchers used a high-speed industrial camera sensor like the ones used in testing vehicles for crash safety. However, the field of view for a part of a cell is astonishingly tiny as compared to that of a car. At high enough frame rates, very few photons hit the sensor, creating a highly noisy image. To overcome this challenge, they coupled an image intensifier with the sensor. After the sensor converts the photons into electrons, the intensifier amplifies electrons to a level that noise from the camera is relatively negligible.

At 10,000 frames per second, they could precisely locate structures within 20 nanometers of their position. This is equivalent to achieving hair-breadth precision at 60 frames per rate, the usual frame rate for live sports broadcasts, except it’s too dark to see anything.

Over two decades back, Fujiwara and Akihiro Kusumi, who is also the corresponding author of this paper, demonstrated that phospholipids and membrane proteins move in a non-random fashion termed hop diffusion. Looking for a visual proof of the mechanism, the authors trained their new imaging method on the cell membranes of epithelial cells. They observed the membrane molecules hop from one compartment of the cell to another. Going beyond existing methods, they were also able to visualize hop diffusion in the cells attached to the substrate.

In an accompanying paper, Fujiwara and colleagues use the ultrafast imaging method to study focal adhesions, molecular structures that helps cells bind to the extracellular matrix around them. With its high frame rate, the method shortened the time required to picture these structures from a few minutes to a few seconds. This allowed the team to study them in living cells as compared to other imaging techniques that work with fixed dead cells.

They found that membrane proteins are loosely clustered in islands. Further, integrin, a transmembrane protein, diffuse in and out of focal adhesions. Both the arrangement of the protein islands and diffusion of integrin allows the latter’s rapid recruitment or removal of integrins for the formation or breakdown of focal adhesions, respectively.

Dynamic interactions between molecules underlie many biological processes. Cells constantly take in information of all kinds to respond quickly to their environment. Ultrafast imaging provides a way to freeze this action in slow motion and will enable deeper insights into the process running from protein folding and gene regulation to transport organelles.

For example, a better understanding of cell membrane dynamics could provide clinically relevant insights. “In cancer metastasis or during development, the cells have to move with each other to form tissues. Understanding focal adhesion is very important,” Fujiwara added.

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Source: Kyoto University ; Yipei Lieu/ Asian Scientist Magaizne