Time-deterministic cryo-optical microscopy developed by University of Osaka — Cells under observation instantly cryofixed

A research group led by PhD student Kosuke Tsuji, Specially Appointed Associate Professor Masahito Yamanaka, and Professor Katsumasa Fujita from the Graduate School of Engineering and Associate Professor Yasuaki Kumamoto from the Institute for Open and Transdisciplinary Research Initiatives, in collaboration with the Immunology Frontier Research Center and the Institute of Scientific and Industrial Research (all part of the University of Osaka), Kyoto Prefectural University of Medicine, and their collaborators, has succeeded in developing time-deterministic cryo-optical microscopy that enables cryofixation of cells under optical microscope observation at arbitrary timing with millisecond-level temporal precision, allowing detailed observation of these cells. By capturing spatio-temporal information about cellular dynamics regarding "what phenomena were occurring at selected timepoints" within cells up until just before cryofixation while visualizing the state of cellular dynamics at "selected timepoints" with high quantitative accuracy and spatial resolution, this technique is expected to contribute broadly to research in life sciences and medicine based on optical microscopy observation of biological samples. The results were published in Light: Science & Applications.

The research group developed an easy-to-handle sample freezing chamber that enables rapid cryofixation of cells during optical microscope observation and detailed observation of samples in their frozen state. In the chamber developed by the group, a mixture of liquid isopentane and propane (approximately -185℃) is injected to rapidly cryofix the sample after reducing the amount of buffer solution around cells to ensure sufficient freezing speed. The cryogen mixture injected remains on the sample, maintaining low temperature even after rapid freezing. The chamber can be easily attached to various commercially available optical microscopes, and sample exchange can be performed in about one minute using spare chamber parts. Furthermore, the group developed a cryogen injection device that injects the cryogen mixture onto samples with ±10 millisecond precision through electric control.

In this research, using the sample chamber they had developed, the group succeeded in instant cryofixation of calcium ions propagating rapidly within cells and organelles moving dynamically inside cells. After freezing the calcium ion distribution, they also succeeded in significantly improving the signal-to-noise ratio through long exposure times, demonstrating that cells can be observed with high quantitative accuracy after freezing.

They also succeeded in two-color super-resolution observation of intracellular calcium ion distribution and actin filaments, as well as three-dimensional super-resolution observation of intracellular calcium ion distribution, demonstrating that "selected timepoints" of cells can be visualized even with observation techniques requiring relatively long exposure times. Furthermore, utilizing optical stimulation techniques for cells and the electrically controlled cryogen injection device, they demonstrated that the time from the initiation of intracellular phenomena to cryofixation can be determined arbitrarily with ±10 millisecond precision.

In addition to these achievements, they observed frozen samples using super-resolution fluorescence microscopy and Raman microscopy. They demonstrated that even with multiple optical imaging techniques requiring different observation times (super-resolution fluorescence microscopy imaging time: 750 milliseconds, Raman microscopy imaging time: 25 minutes), the cellular state at the same time point can be visualized and analyzed. The research group named this technique "time-deterministic cryo-optical microscopy."

Fujita commented: "This research started from a bold change in thinking—'stopping movement to observe' cellular dynamics, without being constrained by the temporal limitations that were challenges in live-cell imaging. The distinctive feature of this technology is its ability to capture the spatio-temporal information of cells while freezing momentary states, enabling detailed observation with high quantitative accuracy using various optical microscopy techniques. I expect this to become one of the fundamental technologies that brings new perspectives to research in biology and medicine."

Journal Information
Publication: Light: Science & Applications
Title: Time-deterministic cryo-optical microscopy
DOI: 10.1038/s41377-025-01941-8