Data availability
The datasets generated and/or analysed during the current study are available in the GenBank/EMBL/DDBJ repositories, accession numbers LC890396 and LC890397.
References
-
Foissner, W. An update of ‘basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa’. Int. J. Syst. Evol. Microbiol. 64, 271–292. https://doi.org/10.1099/ijs.0.057893-0 (2014).
-
Hoshina, R., Tsukii, Y., Harumoto, T. & Suzaki, T. Characterization of a green Stentor with symbiotic algae growing in an extremely oligotrophic environment and storing large amounts of starch granules in its cytoplasm. Sci. Rep. 11, 2865. https://doi.org/10.1038/s41598-021-82416-9 (2020).
-
Ishida, H., Matsumoto, C., Shimada, M. & Suzaki, T. Use of water freeze-drying to prepare unfixed ciliates for scanning electron microscopy. J. Electr. Microsc Tech. Med. Biol. 35, 1–7 (2022).
-
Ishida, H. et al. Method of Preparing unfixed ciliates for scanning electron microscopy without noticeable artifacts. Front. Protistol. 1, 1293531. https://doi.org/10.3389/frpro.2023.1293531 (2023).
-
King, M. V. Dimensional changes in cells and tissues during specimen Preparation for the electron microscope. Cell. Biophys. 18, 31–55. https://doi.org/10.1007/BF02990514 (1991).
-
Billings-Gagliardi, S., Pockwinse, S. M. & Schneider, G. B. Morphological changes in isolated lymphocytes during Preparation for SEM: freeze drying versus critical-point drying. Am. J. Anat. 152, 383–389. https://doi.org/10.1002/aja.1001520308 (1978).
-
Ishida, H., Matsumoto, C., Shimada, M. & Suzaki, T. SEM observation of non-fixed and water freeze-dried Spirostomum ambiguum. Eur. J. Protistol. 85, 125896. https://doi.org/10.1016/j.ejop.2022.125896 (2022).
-
Wibowo, E., Ulya, N., Farizi, M. R. & Fitriyanti, N. Derivation of newton’s law of cooling and heating: heating the water then cooling it down naturally to the room temperature. Momentum Phys. Educ. J. 7, 78–92. https://doi.org/10.21067/mpej.v7i1.6889 (2023).
-
Vollmer, M. Newton’s law of cooling revisited. Eur. J. Phys. 30, 1063–1084. https://doi.org/10.1088/0143-0807/30/5/014 (2009).
-
Zheng, O., Zhang, L., Sun, Q. & Liu, S. Basic theory of ice crystallization based on water molecular structure and ice structure. Foods 13, 2773. https://doi.org/10.3390/foods13172773 (2024).
-
Yan, L., Wang, Z., Liu, C. & Wu, Y. On the determination and simulation of seawater freezing point temperature under high pressure. Adv. Polar Sci. 30, 391–398. https://doi.org/10.13679/j.advps.2019.0018 (2019).
-
Studer, D., Humbel, B. M. & Chiquet, M. Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem. Cell. Biol. 130, 877–889. https://doi.org/10.1007/s00418-008-0500-1 (2008).
-
Song, C. & Suzaki, T. Improved preservation of organelles in Paramecium bursaria by freeze-substitution with glutaraldehyde and osmium tetroxide. J. Electr. Microsc Tech. Med. Biol. 27, 1–8 (2013).
-
Gallo, P. et al. Advances in the study of supercooled water. Eur. Phys. J. E. 44, 143. https://doi.org/10.1140/epje/s10189-021-00139-1 (2021).
-
Kobayashi, M., Sugiura, M., Iwasaki, S., Iwabe, N. & Harumoto, T. Mating pheromone (gamone 1) in Blepharisma: A glycoprotein responsible for species diversity in unicellular organisms (Alveolata, Ciliophora). Microorganisms 12, 299. https://doi.org/10.3390/microorganisms12020299 (2024).
-
Aonuma, H., Naniwa, K., Sugimoto, Y., Ohkawara, K. & Kagaya, K. Embodied latch mechanism of the mandible to power at ultra-high speed in the trap-jaw ant Odontomachus Kuroiwae. J. Exp. Biol. 226, jeb245396. https://doi.org/10.1242/jeb.245396 (2023).
-
Song, C., Murata, K. & Suzaki, T. Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics. Sci. Rep. 7, 1221. https://doi.org/10.1038/s41598-017-01331-0 (2017).
Acknowledgements
We thank the Japanese Red Cross Society for providing human erythrocytes. We would like to thank Editage (www.editage.jp) for English language editing.
Funding
This work was supported by JSPS KAKENHI Grant Number JP21K19168, JP22K06377, JP24KJ1708.
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Ishida, H., Fukuda, N., Shimada, M. et al. Artifact-free preparation of biological samples for SEM by optimized water freeze-drying. Sci Rep (2025). https://doi.org/10.1038/s41598-025-30335-4
-
Received:
-
Accepted:
-
Published:
-
DOI: https://doi.org/10.1038/s41598-025-30335-4
