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A novel approach for 3D reconstruction of mice full-grown oocytes by time-of-flight secondary ion mass spectrometry

  • Alexander Gulin
  • Victor Nadtochenko
  • Alyona Solodina
  • Maria Pogorelova
  • Artem Panait
  • Alexander PogorelovEmail author
Research Paper

Abstract

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell—fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as “nucleolus-like body” (NLB) was detected inside the germinal vesicle in PO3 and CN ions generated by nucleic acids and proteins respectively. Significant difference in PO3 intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3 and CN ion images is 12.9 ± 0.2 μm and 11.9 ± 0.2 μm respectively, which suggests that bright halo of PO3 ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0–2.5 μm size inside nucleoplasm with increased PO3 and CN signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes.

Keywords

ToF-SIMS Single cell imaging Germinal vesicle Nuclear bodies Sputter depth profiling Cell sectioning 

Notes

Acknowledgments

Investigations were performed using the facilities of Semenov FRCCP RAS CCE (no. 506694). We thank A. Astafiev for the help with manuscript revision.

Funding information

This work was supported by RFBR grant 19-53-52007 and FRCCP RAS state task (АААА-А19-119012990175-9) in the part of ToF-SIMS measurements. This work was also supported by the Russian Science Foundation grant 17-76-20014 in the part of bioorganic sample preparation.

Compliance with ethical standards

All animal procedures were approved by the Animal Ethics Committee in Institute of Theoretical and Experimental Biophysics RAS and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2237_MOESM1_ESM.pdf (356 kb)
ESM 1 (PDF 356 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Alexander Gulin
    • 1
    • 2
  • Victor Nadtochenko
    • 1
    • 2
  • Alyona Solodina
    • 1
  • Maria Pogorelova
    • 3
  • Artem Panait
    • 3
  • Alexander Pogorelov
    • 3
    Email author
  1. 1.N.N. Semenov Federal Research Center for Chemical PhysicsRussian Academy of Sciences (FRCCP RAS)MoscowRussian Federation
  2. 2.Department of ChemistryLomonosov Moscow State UniversityMoscowRussian Federation
  3. 3.Institute of Theoretical and Experimental BiophysicsRussian Academy of SciencesPushchinoRussian Federation

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