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


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.


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



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)


  1. 1.
    Denbigh JL, Lockyer NP. ToF-SIMS as a tool for profiling lipids in cancer and other diseases. Mater Sci Technol. 2015;31(2):137–47. Scholar
  2. 2.
    Wehrli PM, Angerer TB, Farewell A, Fletcher JS, Gottfries J. Investigating the role of the stringent response in lipid modifications during the stationary phase in E. coli by direct analysis with time-of-flight-secondary ion mass spectrometry. Anal Chem. 2016;88(17):8680–8. Scholar
  3. 3.
    Tian H, Sparvero LJ, Amoscato AA, Bloom A, Bayir H, Kagan VE, et al. Gas cluster ion beam time-of-flight secondary ion mass spectrometry high-resolution imaging of cardiolipin speciation in the brain: identification of molecular losses after traumatic injury. Anal Chem. 2017;89(8):4611–9. Scholar
  4. 4.
    Urbini M, Petito V, de Notaristefani F, Scaldaferri F, Gasbarrini A, Tortora L. ToF-SIMS and principal component analysis of lipids and amino acids from inflamed and dysplastic human colonic mucosa. Anal Bioanal Chem. 2017;409(26):6097–111. Scholar
  5. 5.
    Yakovleva MA, Gulin AA, Feldman TB, Bel’skich YC, Arbukhanova PM, Astaf’ev AA, et al. Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium. Anal Bioanal Chem. 2016;408(26):7521–8. Scholar
  6. 6.
    Bobrowska J, Moffat J, Awsiuk K, Pabijan J, Rysz J, Budkowski A, et al. Comparing surface properties of melanoma cells using time of flight secondary ions mass spectrometry. Analyst. 2016;141(22):6217–25. Scholar
  7. 7.
    Shon HK, Kim SH, Yoon S, Shin CY, Lee TG. Molecular depth profiling on rat brain tissue sections prepared using different sampling methods. Biointerphases. 2018;13(3). Scholar
  8. 8.
    Bolbach G, Viari A, Galera R, Brunot A, Blais JC. Organic film thickness effect in secondary ion mass-spectrometry and plasma desorption mass-spectrometry. Int J Mass Spectrom Ion Process. 1992;112(1):93–100. Scholar
  9. 9.
    Chandra S, Tjarks W, Lorey DR, Barth RF. Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS). J Microsc (Oxford, U K). 2008;229(1):92–103. Scholar
  10. 10.
    Lanekoff I, Sjovall P, Ewing AG. Relative quantification of phospholipid accumulation in the PC12 cell plasma membrane following phospholipid incubation using TOF-SIMS imaging. Anal Chem. 2011;83(13):5337–43. Scholar
  11. 11.
    Draude F, Korsgen M, Pelster A, Schwerdtle T, Muthing J, Arlinghaus HF. Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS. Anal Bioanal Chem. 2015;407(8):2203–11. Scholar
  12. 12.
    Gulin A, Nadtochenko V, Astafiev A, Pogorelova V, Rtimi S, Pogorelov A. Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes. Analyst. 2016;141(13):4121–9. Scholar
  13. 13.
    Robinson MA, Castner DG. Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis. Biointerphases. 2013;8. Scholar
  14. 14.
    Schaepe K, Kokesch-Himmelreich J, Rohnke M, Wagner AS, Schaaf T, Wenisch S et al. Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS. Biointerphases. 2015;10(1). Scholar
  15. 15.
    Bobrowska J, Pabijan J, Wiltowska-Zuber J, Jany BR, Krok F, Awsiuk K, et al. Protocol of single cells preparation for time of flight secondary ion mass spectrometry. Anal Biochem. 2016;511:52–60. Scholar
  16. 16.
    Rangarajan S, Tyler BJ. Topography in secondary ion mass spectroscopy images. J Vac Sci Technol A. 2006;24(5):1730–6. Scholar
  17. 17.
    Lee JLS, Gilmore IS, Seah MP, Fletcher IW. Topography and field effects in secondary ion mass spectrometry - part I: conducting samples. J Am Soc Mass Spectrom. 2011;22(10):1718–28. Scholar
  18. 18.
    Lee JLS, Gilmore IS, Seah MP, Levick AP, Shard AG. Topography and field effects in secondary ion mass spectrometry Part II: insulating samples. Surf Interface Anal. 2012;44(2):238–45. Scholar
  19. 19.
    Kita NT, Ushikubo T, Fu B, Valley JW. High precision SIMS oxygen isotope analysis and the effect of sample topography. Chem Geol. 2009;264(1-4):43–57. Scholar
  20. 20.
    Vickerman JC. Molecular imaging and depth profiling by mass spectrometry-SIMS, MALDI or DESI? Analyst. 2011;136(11):2199–217. Scholar
  21. 21.
    Seeley EH, Caprioli RM. 3D imaging by mass spectrometry: a new frontier. Anal Chem. 2012;84(5):2105–10. Scholar
  22. 22.
    Fornai L, Angelini A, Klinkert I, Giskes F, Kiss A, Eijkel G, et al. Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging. Anal Bioanal Chem. 2012;404(10):2927–38. Scholar
  23. 23.
    Fletcher JS, Lockyer NP, Vaidyanathan S, Vickerman JC. TOF-SIMS 3D biomolecular imaging of Xenopus laevis oocytes using buckminsterfullerene C60 primary ions. Anal Chem. 2007;79(6):2199–206. Scholar
  24. 24.
    Breitenstein D, Rommel CE, Mollers R, Wegener J, Hagenhoff B. The chemical composition of animal cells and their intracellular compartments reconstructed from 3D mass spectrometry. Angew Chem Int Ed. 2007;46(28):5332–5. Scholar
  25. 25.
    Fletcher JS, Rabbani S, Henderson A, Lockyer NP, Vickerman JC. Three-dimensional mass spectral imaging of HeLa-M cells - sample preparation, data interpretation and visualisation. Rapid Commun Mass Spectrom. 2011;25(7):925–32. Scholar
  26. 26.
    Brison J, Robinson MA, Benoit DS, Muramoto S, Stayton PS, Castner DG. TOF-SIMS 3D imaging of native and non-native species within HeLa cells. Anal Chem. 2013;85(22):10869–77. Scholar
  27. 27.
    Ghosal S, Fallon SJ, Leighton TJ, Wheeler KE, Kristo MJ, Nutcheon ID, et al. Imaging and 3D elemental characterization of intact bacterial spores by high-resolution secondary ion mass spectrometry. Anal Chem. 2008;80(15):5986–92. Scholar
  28. 28.
    Passarelli MK, Ewing AG, Winograd N. Single-cell lipidomics: characterizing and imaging lipids on the sof individual Aplysia californica neurons with cluster secondary ion mass spectrometry. Anal Chem. 2013;85(4):2231–8. Scholar
  29. 29.
    Tian H, Fletcher JS, Thuret R, Henderson A, Papalopulu N, Vickerman JC, et al. Spatiotemporal lipid profiling during early embryo development of Xenopus laevis using dynamic ToF-SIMS imaging. J Lipid Res. 2014;55(9):1970–80. Scholar
  30. 30.
    Piwowar AM, Keskin S, Delgado MO, Shen K, Hue JJ, Lanekoff I, et al. C60-ToF SIMS imaging of frozen hydrated HeLa cells. Surf Interface Anal. 2013;45(1):302–4. Scholar
  31. 31.
    Sheng LF, Cai LS, Wang J, Li ZP, Mo YX, Zhang SC, et al. Simultaneous imaging of newly synthesized proteins and lipids in single cell by TOF-SIMS. Int J Mass Spectrom. 2017;421:238–44. Scholar
  32. 32.
    Pavlyukov MS, Gulin AA, Astafiev AA, Svetlichny VY, Gularyan SK. Lateral heterogeneity of cholesterol distribution in cell plasma membrane: investigation by microfluorimetry, immunofluorescence, and TOF-SIMS. Biochem (Mosc) Suppl Ser A Membr Cell Biol. 2019;13(1):50–7. Scholar
  33. 33.
    Astafiev AA, Gulin AA, Osychenko AA, Solodina AE, Syrchina MS, Titov AA, et al. Structural features of the nucleolus in the mouse germinal vesicle oocyte revealed by AFM, SEM, and ToF-SIMS. Nanotechnol Russ. 2017;12(7-8):444–7. Scholar
  34. 34.
    Kresoja-Rakic J, Santoro R. Nucleolus and rRNA gene chromatin in early embryo development. Trends Genet. 2019. Scholar
  35. 35.
    Fulka H, Langerova A. Nucleoli in embryos: a central structural platform for embryonic chromatin remodeling? Chromosom Res. 2019;27(1-2):129–40. Scholar
  36. 36.
    Sanni OD, Wagner MS, Briggs D, Castner DG, Vickerman JC. Classification of adsorbed protein static ToF-SIMS spectra by principal component analysis and neural networks. Surf Interface Anal. 2002;33(9):715–28. Scholar
  37. 37.
    Shishova KV, Lavrentyeva EA, Dobrucki JW, Zatsepina OV. Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev Biol. 2015;397(2):267–81. Scholar
  38. 38.
    Ingram MJ, Hogben CAM. Procedure for the study of biological soft tissue with the electron microprobe. Dev Appl Spectrosc. 1968;5:43–54.CrossRefGoogle Scholar
  39. 39.
    Malm J, Giannaras D, Riehle MO, Gadegaard N, Sjovall P. Fixation and drying protocols for the preparation of cell samples for time-of-flight secondary ion mass spectrometry analysis. Anal Chem. 2009;81(17):7197–205. Scholar
  40. 40.
    Pogorelov AG, Pogorelova VN. Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction. J Microsc (Oxford, U K). 2008;232(1):36–43. Scholar
  41. 41.
    Pogorelov AG, Kantor GM, Sakharova NI, Smirnov AA, Aksirov AM, Chaǐlakhian LM. 3-D reconstruction of early preimplantation mouse embryo. Tsitologiia. 2005;47(8):686–91.PubMedGoogle Scholar
  42. 42.
    Efimov AE, Tonevitsky AG, Dittrich M, Matsko NB. Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples. J Microsc (Oxford, U K). 2007;226(3):207–17. Scholar
  43. 43.
    Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. 2017;18(5):285–98. Scholar
  44. 44.
    Shin Y, Chang YC, Lee DSW, Berry J, Sanders DW, Ronceray P, et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell. 2018;175(6):1481-+. Scholar
  45. 45.
    Schuh M, Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell. 2007;130(3):484–98. Scholar
  46. 46.
    Bonnet-Garnier A, Feuerstein P, Chebrout M, Fleurot R, Jan HU, Debey P, et al. Genome organization and epigenetic marks in mouse germinal vesicle oocytes. Int J Dev Biol. 2012;56(10-12):877–87. Scholar
  47. 47.
    Fletcher JS. Latest applications of 3D ToF-SIMS bio-imaging. Biointerphases. 2015;10(1). Scholar
  48. 48.
    Hagenhoff B, Breitenstein D, Tallarek E, Mollers R, Niehuis E, Sperber M, et al. Detection of micro- and nano-particles in animal cells by ToF-SIMS 3D analysis. Surf Interface Anal. 2013;45(1):315–9. Scholar
  49. 49.
    Wucher A, Cheng J, Winograd N. Protocols for three-dimensional molecular imaging using mass spectrometry. Anal Chem. 2007;79(15):5529–39. Scholar
  50. 50.
    Robinson MA, Graham DJ, Castner DG. ToF-SIMS depth profiling of cells: z-correction, 3D imaging, and sputter rate of individual NIH/3T3 fibroblasts. Anal Chem. 2012;84(11):4880–5. Scholar
  51. 51.
    Patkin AJ, Chandra S, Morrison GH. Differential sputtering correction for ion microscopy with image depth profiling. Anal Chem. 1982;54(14):2507–10. Scholar
  52. 52.
    Wehbe N, Tabarrant T, Brison J, Mouhib T, Delcorte A, Bertrand P, et al. TOF-SIMS depth profiling of multilayer amino-acid films using large argon cluster Ar-n(+), C-60(+) and Cs+ sputtering ions: a comparative study. Surf Interface Anal. 2013;45(1):178–80. Scholar
  53. 53.
    Sjovall P, Rading D, Ray S, Yang L, Shard AG. Sample cooling or rotation improves C-60 organic depth profiles of multilayered reference samples: results from a VAMAS interlaboratory study. J Phys Chem B. 2010;114(2):769–74. Scholar
  54. 54.
    Seki T, Matsuo J. Surface smoothing with large current cluster ion beam. Nucl Instrum Meth B. 2004;216:191–5. Scholar
  55. 55.
    Ichiki K, Ninomiya S, Nakata Y, Yamada H, Seki T, Aoki T, et al. Surface morphology of PMMA surfaces bombarded with size-selected gas cluster ion beams. Surf Interface Anal. 2011;43(1-2):120–2. Scholar

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

Personalised recommendations