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Applying the Expansion Microscopy Method in Neurobiology

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Abstract

Many biological studies require the analysis of ultrastructural changes at the level of cell organelles and macromolecules. Since the resolution of modern confocal microscope is limited by the diffraction limit (200–300 nm), it is impossible to study such small objects using standard fluorescence microscopy. Ultra-high resolution microscopy methods require expensive equipment and are technically difficult in use, which in turn limits their widespread practical application. However, recently appeared methods make it possible to increase the resolution of microscopy not by improving the image registration system, but by physically isotropic expansion of a biological sample using a controlled chemical process. Due to this method, called expansion or expansive microscopy (ExM), it became possible to obtain three-dimensional images of samples with a resolution sufficient to study individual cell organelles using a conventional confocal microscope. This review covers the history of this method, its basic principles and examples of use in various fields of biology and medicine, as well as reflects future directions for improving this technology. The article discusses the methodological features of the ExM application in a study of brain tissue samples using the algorithm that allows adaptation of the standard protocol to the goals and objectives of a particular study.

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REFERENCES

  1. What Is a Polymer? Live Science. https://www.livescience.com/60682-polymers.html

  2. Tanaka T (1978) Collapse of Gels and the Critical Endpoint. Phys Rev Lett 40:820–823. https://doi.org/10.1103/PhysRevLett.40.820

    Article  CAS  Google Scholar 

  3. Chen F, Tillberg PW, Boyden ES (2015) Expansion microscopy. Science (80) 347:543–548. https://doi.org/10.1126/science.1260088

    Article  CAS  Google Scholar 

  4. Lezhnev EI, Popova II, Kuzmin SV, Slashchev SM (2001). Confocal laser scanning microscopy: principles, device, application (part 2). Scientific Instrumentation 11 (3):26-42 (In Russ).

    Google Scholar 

  5. Biomembranes. Molecular structure and function (ed R Gennis) (In Russ). ISBN 5-03-002419-0

  6. Chen F, Wassie AT, Cote AJ, Sinha A, Alon S, Asano S, Daugharthy ER, Chang JB, Marblestone A, Church GM, Raj A, Boyden ES (2016) Nanoscale imaging of RNA with expansion microscopy. Nat Methods 13:679–684. https://doi.org/10.1038/nmeth.3899

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhao Y, Bucur O, Irshad H, Chen F, Weins A, Stancu AL, Oh EY, Distasio M, Torous V, Glass B, Stillman IE, Schnitt SJ, Beck AH, Boyden ES (2017) Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat Biotechnol 35:757–764. https://doi.org/10.1038/nbt.3892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chozinski TJ, Halpern AR, Okawa H, Kim HJ, Tremel GJ, Wong ROL, Vaughan JC (2016) Expansion microscopy with conventional antibodies and fluorescent proteins. Nat Methods 13:485–488. https://doi.org/10.1038/nmeth.3833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chang JB, Chen F, Yoon YG, Jung EE, Babcock H, Kang JS, Asano S, Suk HJ, Pak N, Tillberg PW, Wassie AT, Cai D, Boyden ES (2017) Iterative expansion microscopy. Nat Methods 14:593–599. https://doi.org/10.1038/nmeth.4261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tillberg PW, Chen F, Piatkevich KD, Zhao Y, Yu CC, English BP, Gao L, Martorell A, Suk HJ, Yoshida F, Degennaro EM, Roossien DH, Gong G, Seneviratne U, Tannenbaum SR, Desimone R, Cai D, Boyden ES (2016) Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34:987–992. https://doi.org/10.1038/nbt.3625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO (2018) X10 expansion microscopy enables 25—nm resolution on conventional microscopes. EMBO Rep 19:(9):e45836. https://doi.org/10.15252/embr.201845836

    Article  CAS  Google Scholar 

  12. Cipriano BH, Banik SJ, Sharma R, Rumore D, Hwang W, Briber RM, Raghavan SR (2014) Superabsorbent hydrogels that are robust and highly stretchable. Macromolecules 47:4445–4452. https://doi.org/10.1021/ma500882n

    Article  CAS  Google Scholar 

  13. Cahoon CK, Yu Z, Wang Y, Guo F, Unruh JR, Slaughter BD, Hawley RS (2017) Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc Natl Acad Sci USA 114:E6857–E6866. https://doi.org/10.1073/pnas.1705623114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Halpern AR, Alas GCM, Chozinski TJ, Paredez AR, Vaughan JC (2017) Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization. ACS Nano 11:12677–12686. https://doi.org/10.1021/acsnano.7b07200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gao M, Maraspini R, Beutel O, Zehtabian A, Eickholt B, Honigmann A, Ewers H (2018) Expansion Stimulated Emission Depletion Microscopy (ExSTED). ACS Nano 12:4178–4185. https://doi.org/10.1021/acsnano.8b00776

    Article  CAS  PubMed  Google Scholar 

  16. Gambarotto D, Zwettler FU, Le Guennec M, Schmidt-Cernohorska M, Fortun D, Borgers S, Heine J, Schloetel JG, Reuss M, Unser M, Boyden ES, Sauer M, Hamel V, Guichard P (2019) Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat Methods 16:71–74. https://doi.org/10.1038/s41592-018-0238-1

    Article  CAS  PubMed  Google Scholar 

  17. Asano SM, Gao R, Wassie AT, Tillberg PW, Chen F, Boyden ES (2018) Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues. Curr Protoc Cell Biol 80(1):e56. https://doi.org/10.1002/cpcb.56

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Crittenden JR, Tillberg PW, Riad MH, Shima Y, Gerfen CR, Curry J, Housman DE, Nelson SB, Boyden ES, Graybiel AM (2016) Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons. Proc Natl Acad Sci U S A 113:11318–11323. https://doi.org/10.1073/pnas.1613337113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mosca TJ, Luginbuhl DJ, Wang IE, Luo L (2017) Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. Elife 6:27347. https://doi.org/10.7554/eLife.27347

    Article  Google Scholar 

  20. Freifeld L, Odstrcil I, Förster D, Ramirez A, Gagnon JA, Randlett O, Costa EK, Asano S, Celiker OT, Gao R, Martin-Alarcon DA, Reginato P, Dick C, Chen L, Schoppik D, Engert F, Baier H, Boyden ES (2017) Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proc Natl Acad Sci U S A 114:E10799–E10808. https://doi.org/10.1073/pnas.1706281114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Deshpande T, Li T, Herde MK, Becker A, Vatter H, Schwarz MK, Henneberger C, Steinhäuser C, Bedner P (2017) Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy. Glia 65:1809–1820. https://doi.org/10.1002/glia.23196

    Article  PubMed  Google Scholar 

  22. Wang IE, Lapan SW, Scimone ML, Clandinin TR, Reddien PW (2016) Hedgehog signaling regulates gene expression in planarian glia. Elife 5:e16996. https://doi.org/10.7554/eLife.16996

    Article  PubMed  PubMed Central  Google Scholar 

  23. Truckenbrodt S, Sommer C, Rizzoli SO, Danzl JG (2019) A practical guide to optimization in X10 expansion microscopy. Nat Protoc 14:832–863. https://doi.org/10.1038/s41596-018-0117-3

    Article  CAS  PubMed  Google Scholar 

  24. Gao R, Yu CC (Jay), Gao L, Piatkevich KD, Neve RL, Upadhyayula S, Boyden ES (2019) A highly homogeneous expansion microscopy polymer composed of tetrahedron-like monomers. bioRxiv 814111

  25. Shi X, Li Q, Dai Z, Tran AA, Feng S, Ramirez AD, Lin Z, Wang X, Chow TT, Seiple IB, Huang B (2019) Label-retention expansion microscopy. bioRxiv 687954

  26. Sun D, Fan X, Shi Y, Zhang H, Huang Z, Cheng B, Tang Q, Li W, Zhu Y, Bai J, Liu W, Li Y, Wang X, Lei X, Chen X (2021) Click-ExM enables expansion microscopy for all biomolecules. Nat Methods 18:107–113. https://doi.org/10.1038/s41592-020-01005-2

    Article  CAS  PubMed  Google Scholar 

  27. Wang Y, Yu Z, Cahoon CK, Parmely T, Thomas N, Unruh JR, Slaughter BD, Hawley RS (2018) Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes. Nat Protoc 13:1869–1895. https://doi.org/10.1038/s41596-018-0023-8

    Article  CAS  PubMed  Google Scholar 

  28. Gambarotto D, Zwettler FU, Cernohorska M, Fortun D, Borgers S, Heine J, Schloetel JG, Reuss M, Unser M, Boyden ES, Sauer M, Hamel V, Guichard P (2018) Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM). bioRxiv 308270

  29. Tong Z, Beuzer P, Ye Q, Axelrod J, Hong Z, Cang H (2016) Ex-STORM: Expansion Single Molecule Super-resolution Microscopy. https://doi.org/10.1101/049403

    Book  Google Scholar 

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Funding

This work was supported by the Russian Science Foundation; grant No. 20–45–01004 to I.B. Bezprozvanny. E.I. Pchitskaya is a President of the Russian Federation scholarship recipient; No. SP–3122.2021.4.

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Authors and Affiliations

  1. Institute of Biomedical Systems and Biotechnology, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia

    K. Z. Derevtsova, E. I. Pchitskaya, A. V. Rakovskaya & I. B. Bezprozvanny

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  1. K. Z. Derevtsova
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  2. E. I. Pchitskaya
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  3. A. V. Rakovskaya
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  4. I. B. Bezprozvanny
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Contributions

The idea of work and the planning of the experiment (I.B.B., E.I.P., K.Z.D.), data collection (K.Z.D., E.I.P., A.V.R.), data processing (K.Z.D., E.I.P., A.V.R.), writing and editing the manuscript (K.Z.D., E.I.P., I.B.B.).

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Correspondence to K. Z. Derevtsova.

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CONFLICT OF INTEREST

The authors declare that they have no evident or potential conflict of interest related to the publication of this article.

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Translated by A. Polyanovsky

Russian Text © The Author(s), 2021, published in Rossiiskii Fiziologicheskii Zhurnal imeni I.M. Sechenova, 2021, Vol. 107, Nos. 4–5, pp. 568–583https://doi.org/10.31857/S0869813921040075.

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Derevtsova, K.Z., Pchitskaya, E.I., Rakovskaya, A.V. et al. Applying the Expansion Microscopy Method in Neurobiology. J Evol Biochem Phys 57, 681–693 (2021). https://doi.org/10.1134/S0022093021030157

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