Time-deterministic cryo-optical microscopy
Fluorescence microscopy enables the visualization of cellular morphology, molecular distribution, ion distribution, and their dynamic behaviors during biological processes. Enhancing the signal-to-noise ratio (SNR) in fluorescence imaging improves the quantification accuracy and spatial resolution; however, achieving high SNR at fast image acquisition rates, which is often required to observe cellular dynamics, still remains a challenge. In this study, we developed a technique to rapidly freeze biological cells in milliseconds during optical microscopy observation. Compared to chemical fixation, rapid freezing provides rapid immobilization of samples while more effectively preserving the morphology and conditions of cells. This technique combines the advantages of both live-cell and cryofixation microscopy, i.e., temporal dynamics and high SNR snapshots of selected moments, and is demonstrated by fluorescence and Raman microscopy with high spatial resolution and quantification under low temperature conditions. Furthermore, we also demonstrated that intracellular calcium dynamics can be frozen rapidly and visualized using fluorescent ion indicators, suggesting that ion distribution and conformation of the probe molecules can be fixed both spatially and temporally. These results confirmed that our technique can time-deterministically suspend and visualize cellular dynamics while preserving molecular and ionic states, indicating the potential to provide detailed insights into sample dynamics with improved spatial resolution and temporal accuracy in observations.
Authors
- Yoshinori Harada (ORCID: https://orcid.org/0000-0001-5942-130X)
- Meng Carla Wang (ORCID: https://orcid.org/0000-0002-5898-6007)
- Yuki Tanabe (ORCID: https://orcid.org/0000-0001-6782-888X)
- Shoko Tamura
- Katsumasa Fujita (ORCID: https://orcid.org/0000-0002-2284-375X)
- Masahito Yamanaka (ORCID: https://orcid.org/0000-0002-5862-177X)
- Shun‐ichi Fukushima
- Nicholas I. Smith (ORCID: https://orcid.org/0000-0002-7835-199X)
- Takeharu Nagai (ORCID: https://orcid.org/0000-0003-2650-9895)
- Kentaro Mochizuki (ORCID: https://orcid.org/0009-0006-2015-1457)
- Kazuhiro Tsuji
- Y Kumamoto (ORCID: https://orcid.org/0000-0002-4398-288X)
- Zhiheng Yu (ORCID: https://orcid.org/0000-0002-7114-2570)
- Rainer Heintzmann (ORCID: https://orcid.org/0000-0002-4950-1936)
- Momoko Shiozaki (ORCID: https://orcid.org/0000-0002-5640-8959)
- Hideo Tanaka (ORCID: https://orcid.org/0000-0002-7297-0183)
- Wakana Miyamura
- Xiaowei Zhao (ORCID: https://orcid.org/0000-0002-9868-5102)
- Toshiki Kubo (ORCID: https://orcid.org/0000-0002-5696-4111)
- Kenta Mizushima (ORCID: https://orcid.org/0009-0002-8806-0798)
- Hanae Hirano
- Kentaro Nishida (ORCID: https://orcid.org/0000-0002-4540-3319)
- Kazunori Sugiura
- K. Kono
- Takumi Kunimoto
- Heqi Xi
Institutions
- Howard Hughes Medical Institute (US)
- Osaka University of Economics (JP)
- Janelia Research Campus (US)
- The Open University of Japan (JP)
- Leibniz Institute of Photonic Technology (DE)
- Ube Frontier University (JP)
- Kyoto Prefectural University of Medicine (JP)
- Osaka Health Science University (JP)
- Friedrich Schiller University Jena (DE)
- The University of Osaka (JP)
Publication Details
- Journal
- Light Science & Applications
- Published
- 2025-08-22
- DOI
- https://doi.org/10.1038/s41377-025-01941-8
- Citations
- 3
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 4.90
Funders
- Howard Hughes Medical Institute
- National Institute of Advanced Industrial Science and Technology
- Deutsche Forschungsgemeinschaft
- Leibniz-Gemeinschaft
- Japan Science and Technology Agency
- Core Research for Evolutional Science and Technology
- Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie – Hans-Knöll-Institut
- Co-creation place formation support program