Rapid freezing of intracellular calcium ion propagation happens in milliseconds. Credit: 2025, Kosuke Tsuji, Masahito Yamanaka et al., Time-deterministic cryo-optical microscopy, Light:Science&Applications
Researchers have observed accurate, high-resolution snapshots of cell activity at a precisely chosen moment using a new cryo-optical microscopy technique.
This novel method offers new possibilities for observing fast, dynamic cellular events, which the authors hope will provide future researchers with a crucial tool to further explore the mechanisms behind key biological processes.
Researchers often use optic microscopy, a type of light microscope, to study essential processes that occur in cells. But the high speed at which these processes occur, alongside the high resolution needed to observe them, often makes these types of studies very difficult.
One of the main challenges of using this method is balancing how much time passes between consecutive measurements with how much light can be collected for the image – known as the ‘photon budget’.
The team from Japan’s University of Osaka overcame this barrier by changing up their method.
“Instead of chasing speed in imaging, we decided to freeze the entire scene,” says Kosuke Tsuji, a lead author of the study.
The research focused on calcium ions moving through live heart muscle cells in a process called calcium ion wave propagation. The team successfully froze the calcium wave at different stages of the process.
“We developed a special sample-freezing chamber to combine the advantages of live-cell and cryo-fixation microscopy. By rapidly freezing live cells under the optical microscope, we could observe a frozen snapshot of the cellular dynamics at high resolutions,” says Tsuji.
The team used UV light stimulation to freeze the ions with 10ms precision and observed them in 3 dimensions using a super-resolution technique. Due to the slow imaging acquisition speed of this technique, it is usually ineffective in capturing fast-moving cells.
(A) Concept of the time-deterministic cryo-optical microscopy technique and (bottom) rapid freezing of intracellular calcium ion wave propagation within milliseconds. (B) On-stage freezing chamber and improved signal-to-noise ratio through extended exposure time after rapid freezing of intracellular calcium ion propagation. Credit: 2025, Kosuke Tsuji, Masahito Yamanaka et al., Time-deterministic cryo-optical microscopy, Light: Science & Applications
The frozen nature of the cell meant that the researchers could use exposure times 1,000 times longer than practical live-cell imaging.
“While cells are immobilised, we can take the opportunity to perform highly accurate quantitative measurements with a variety of optical microscopy tools,” says Masahito Yamanaka, one of the lead authors.
“Our technique preserves both spatial and temporal features of live cells with instantaneous freezing, making it possible to observe their states in detail.”
The almost-instantaneous freezing of the cells also allowed the researchers to combine different imaging techniques more easily.
Throughout the study, the research team was able to successfully combine a variety of techniques – like spontaneous Raman microscopy and super-resolution fluorescence microscopy – on the same frozen cells for more accurate results.
“This research began with a bold shift in perspective: to arrest dynamic cellular processes during optical imaging rather than struggle to track them in motion,” says senior author Katsumasa Fujita.
“We believe this will serve as a powerful foundational technique, offering new insights across life-science and medical research.”
The new cell-imaging method has been published in the journal Light: Science and Applications.