A novel approach for 3D reconstruction of mice full-grown oocytes by time-of-flight secondary ion mass spectrometry
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.
KeywordsToF-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.
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.
- 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. https://doi.org/10.1021/acs.analchem.6b01981.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1021/acs.analchem.7b00164.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1007/s00216-017-0546-9.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1007/s00216-016-9854-8.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1111/j.1365-2818.2007.01869.x.CrossRefGoogle Scholar
- 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). https://doi.org/10.1116/1.4915263.CrossRefGoogle Scholar
- 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. https://doi.org/10.1194/jlr.D048660.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1134/s1990747818040098.CrossRefGoogle Scholar
- 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. https://doi.org/10.1111/j.1365-2818.2007.01773.x.CrossRefGoogle Scholar
- 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. https://doi.org/10.1002/sia.5121.CrossRefGoogle Scholar