Time-deterministic cryo-optical microscopy

Kosuke Tsuji; Masahito Yamanaka; Yasuaki Kumamoto; Shoko Tamura; Wakana Miyamura; Toshiki Kubo; Kenta Mizushima; Kakeru Kono; Hanae Hirano; Momoko Shiozaki; Xiaowei Zhao; Heqi Xi; Kazunori Sugiura; Shun-ichi Fukushima; Takumi Kunimoto; Yoshino Tanabe; Kentaro Nishida; Kentaro Mochizuki; Yoshinori Harada; Nicholas I. Smith; Rainer Heintzmann; Zhiheng Yu; Meng C. Wang; Takeharu Nagai; Hideo Tanaka; Katsumasa Fujita

doi:10.1038/s41377-025-01941-8

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Time-deterministic cryo-optical microscopy

Original Articles

- Time-deterministic cryo-optical microscopy
- Light: Science & Applications Vol. 14, Issue 10, Pages: 3069-3081(2025)
- Affiliations：
  
  1.Department of Applied Physics, Graduate School of Engineering, The University of Osaka, Osaka, Japan
  2.Advanced Photonics and Biosensing Open Innovation Laboratory, AIST-Osaka University, Osaka, Japan
  3.Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research Initiatives, The University of Osaka, Osaka, Japan
  4.Department of Pathology and Cell Regulation, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
  5.Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, The University of Osaka, Osaka, Japan
  6.Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA
  7.Department of Biomolecular Science and Engineering, SANKEN, The University of Osaka, Osaka, Japan
  8.Immunology Frontier Research Center, The University of Osaka, Osaka, Japan
  9.Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University Jena, Jena, Germany
  10.Leibniz Institute of Photonic Technology, Jena, Germany
- Author bio：
  
  Masahito Yamanaka (yamanaka@ap.eng.osaka-u.ac.jp)
  Katsumasa Fujita (fujita@ap.eng.osaka-u.ac.jp)
- Funds：
- DOI：10.1038/s41377-025-01941-8
  CLC：
- Received：21 December 2024，
  
  Revised：2025-06-17，
  
  Accepted：26 June 2025，
  
  Published Online：23 August 2025，
  
  Published：31 October 2025
- Accepted：
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Tsuji, K. et al. Time-deterministic cryo-optical microscopy. Light: Science & Applications, 14, 3069-3081 (2025).
DOI：

Tsuji, K. et al. Time-deterministic cryo-optical microscopy. Light: Science & Applications, 14, 3069-3081 (2025). DOI： 10.1038/s41377-025-01941-8.

摘要

Abstract

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.

关键词

Keywords

references

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