New microscopy technique freeze-frames the cellular world

Optical microscopy is a key technique for understanding dynamic biological processes in cells, but observing these high-speed cellular dynamics accurately, at high spatial resolution, has long been a formidable task. Now researchers from The University of Osaka, together with collaborating institutions, have unveiled a cryo-optical microscopy technique that take a high-resolution, quantitatively accurate snapshot at a precisely selected timepoint in dynamic cellular activity. Their work has been published in the journal Light: Science & Applications.

Capturing fast dynamic cellular events with spatial detail and quantifiability has been a major challenge owing to a fundamental trade-off between temporal resolution and the ‘photon budget’ — that is, how much light can be collected for the image. With limited photons and only dim, noisy images, important features in both space and time become lost in the noise.

“Instead of chasing speed in imaging, we decided to freeze the entire scene,” said Kosuke Tsuji, a lead author on the new study. “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.”

For instance, the team froze calcium ion wave propagation in live heart-muscle cells. The intricately detailed frozen wave was then observed in three dimensions using a super-resolution technique that cannot normally observe fast cellular dynamics due to its slow imaging acquisition speed.

By freezing cells labelled with a fluorescent calcium ion probe, the researchers were able to use exposure times 1000 times longer than practical in live-cell imaging, substantially increasing measurement accuracy. To capture transient biological events at precisely defined moments, they integrated an electrically triggered cryogen injection system. With UV light stimulation to induce calcium ion waves, this system enabled freezing of the calcium ion waves at a specific time point after the initiation of the event, with 10 ms precision — allowing the team to arrest transient biological processes with impressive temporal accuracy.

Finally, the team tuned their attention to combining different imaging techniques, which are often difficult to align in time. By the near-instantaneous freezing of samples, multiple imaging modalities can now be applied sequentially without worrying about temporal mismatch. In their study, the team combined spontaneous Raman microscopy and super-resolution fluorescence microscopy on the same cryofixed cells. This allowed them to view intricate cellular information from a number of perspectives at the exact same point in time.

“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,” said senior author Katsumasa Fujita.

Co-lead author Masahito Yamanaka added, “Our technique preserves both spatial and temporal features of live cells with instantaneous freezing, making it possible to observe their states in detail. While cells are immobilised, we can take the opportunity to perform highly accurate quantitative measurements with a variety of optical microscopy tools.”

This innovation thus opens new avenues for observing fast, transient cellular events, providing researchers with a powerful tool to explore the mechanisms underlying dynamic biological processes.

Top image (cropped video still) and above video: Rapid freezing of intracellular calcium ion propagation in milliseconds. Video credit: 2025, Kosuke Tsuji, Masahito Yamanaka et al., Time-deterministic cryo-optical microscopy, Light: Science & Applications (CC BY 4.0).