Correlative Microscopy of Individual Cells: Sequential Application of Microscopic Systems with Increasing Resolution to Study the Nuclear Landscape

  • Barbara Hübner
  • Thomas Cremer
  • Jürgen Neumann
Part of the Methods in Molecular Biology book series (MIMB, volume 1042)


The term correlative microscopy denotes the sequential visualization of one and the same cell using various microscopic techniques. Correlative microscopy provides a unique platform to combine the particular strength of each microscopic approach and compensate for its specific limitations. As an example, we report results of a correlative microscopic study exploring features of the nuclear landscape in HeLa cells. We present a detailed protocol to first investigate distinct structural features of a living cell in space and time (4D) using spinning disk laser scanning microscopy (SDLSM). Then, after fixation and staining of selected structures (e.g., by means of immunodetection), details of these structures are explored at increasingly higher resolution using three-dimensional (3D) confocal laser scanning microscopy (CLSM); super-resolution fluorescence microscopy, such as three-dimensional structured illumination microscopy (3D-SIM); and transmission electron microscopy (TEM). We discuss problems involved in the comparison of images of a given cell nucleus recorded with different microscopic approaches, which requires not only a compensation for different resolutions but also for various distortions.

Key words

Correlative microscopy Live cell microscopy Super-resolution fluorescence microscopy Transmission electron microscopy Relocalization of cells Nuclear architecture Hypercondensed chromatin HCC 


1 Mbp CDs

Megabase-sized chromatin domains




Three-dimensional structured illumination microscopy




Chromatin compartment


Confocal laser scanning microscopy


Chromosome territory–interchromatin compartment


Electron microscopy


Fetal calf serum


Focused ion beam


Fluorescence in situ hybridization


Fluorescence recovery after photobleaching


Green fluorescent protein


Histone 2B tagged with red fluorescent protein


Histone 3 tri-methylated at lysine 4


Histone 3 tri-methylated at lysine 9


Hypercondensed chromatin


Interchromatin compartment


Optical transfer function


Phosphate buffered saline


1× PBS with 0.02 % Tween




Perichromatin region




Point-spread function




Region of interest


Spinning disk laser scanning microscopy


Scanning electron microscopy


Structured illumination microscopy


Transmission electron microscopy


Wide field


  1. 1.
    Caplan J, Niethammer M, Taylor RM 2nd, Czymmek KJ (2011) The power of correlative microscopy: multi-modal, multi-scale, multi-dimensional. Curr Opin Struct Biol 21(5):686–693PubMedCrossRefGoogle Scholar
  2. 2.
    Giepmans BN (2008) Bridging fluorescence microscopy and electron microscopy. Histochem Cell Biol 130(2):211–217PubMedCrossRefGoogle Scholar
  3. 3.
    Muller-Reichert T, Verkade P (2012) Introduction to correlative light and electron microscopy. Methods Cell Biol 111:xvii–xixPubMedCrossRefGoogle Scholar
  4. 4.
    Svitkina TM, Borisy GG (1998) Correlative light and electron microscopy of the cytoskeleton of cultured cells. Methods Enzymol 298:570–592PubMedCrossRefGoogle Scholar
  5. 5.
    Cremer C, Masters BR (2013) Resolution enhancement techniques in microscopy. Eur Phys J H 38(3):281–344Google Scholar
  6. 6.
    Toomre DK, Langhorst MF, Davidson MW (2012) Introduction to spinning disk confocal microscopy. Accessed 29 Nov 2012
  7. 7.
    Cremer C (2012) Optics far beyond the diffraction limit. In: Träger F (ed) Springer handbook of laser and optics. Springer, New York, pp 1359–1397CrossRefGoogle Scholar
  8. 8.
    Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2):82–87PubMedCrossRefGoogle Scholar
  9. 9.
    Heintzmann R, Cremer C (1998) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE 3568:185–196CrossRefGoogle Scholar
  10. 10.
    Gustafsson MG et al (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94(12):4957–4970PubMedCrossRefGoogle Scholar
  11. 11.
    Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190(2):165–175PubMedCrossRefGoogle Scholar
  12. 12.
    Fiolka R, Shao L, Rego EH, Davidson MW, Gustafsson MG (2012) Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci U S A 109(14):5311–5315PubMedCrossRefGoogle Scholar
  13. 13.
    Shao L, Kner P, Rego EH, Gustafsson MG (2011) Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 8(12):1044–1046PubMedCrossRefGoogle Scholar
  14. 14.
    Jones SA, Shim SH, He J, Zhuang X (2011) Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8(6):499–508PubMedCrossRefGoogle Scholar
  15. 15.
    Vaughan JC, Jia S, Zhuang X (2012) Ultrabright photoactivatable fluorophores created by reductive caging. Nat Methods 9(12):1181–1184PubMedCrossRefGoogle Scholar
  16. 16.
    Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11):e329PubMedCrossRefGoogle Scholar
  17. 17.
    Zankel A, Kraus B, Poelt P, Schaffer M, Ingolic E (2009) Ultramicrotomy in the ESEM, a versatile method for materials and life sciences. J Microsc 233(1):140–148PubMedCrossRefGoogle Scholar
  18. 18.
    Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28(12):2959–2964PubMedCrossRefGoogle Scholar
  19. 19.
    Rouquette J et al (2009) Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: a novel electron-microscopic approach to reconstructing nuclear architecture. Chromosome Res 17(6):801–810PubMedCrossRefGoogle Scholar
  20. 20.
    Villinger C et al (2012) FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells. Histochem Cell Biol 138(4):549–556PubMedCrossRefGoogle Scholar
  21. 21.
    Schroeder-Reiter E, Sanei M, Houben A, Wanner G (2012) Current SEM techniques for de- and re-construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes. J Microsc 246(1):96–106PubMedCrossRefGoogle Scholar
  22. 22.
    Hayat M (2000) Principles and techniques of electron microscopy: biological applications. Cambridge University Press, CambridgeGoogle Scholar
  23. 23.
    Testillano PS et al (1991) A specific ultrastructural method to reveal DNA: the NAMA-Ur. J Histochem Cytochem 39(10):1427–1438PubMedCrossRefGoogle Scholar
  24. 24.
    Vazquez-Nin GH, Biggiogera M, Echeverria OM (1995) Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA. Eur J Histochem 39(2):101–106PubMedGoogle Scholar
  25. 25.
    Dubochet J, Sartori Blanc N (2001) The cell in absence of aggregation artifacts. Micron 32(1):91–99PubMedCrossRefGoogle Scholar
  26. 26.
    Glaeser RM (2008) Cryo-electron microscopy of biological nanostructures. Phys Today 61:48–54CrossRefGoogle Scholar
  27. 27.
    Cremer T, Cremer C (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2(4):292–301PubMedCrossRefGoogle Scholar
  28. 28.
    Cremer T, Cremer M (2010) Chromosome territories. Cold Spring Harb Perspect Biol 2(3):a003889PubMedCrossRefGoogle Scholar
  29. 29.
    Dixon JR et al (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485(7398):376–380PubMedCrossRefGoogle Scholar
  30. 30.
    Lieberman-Aiden E et al (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950):289–293PubMedCrossRefGoogle Scholar
  31. 31.
    Markaki Y et al (2012) The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture. Bioessays 34(5):412–426PubMedCrossRefGoogle Scholar
  32. 32.
    Nora EP et al (2012) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485(7398):381–385PubMedCrossRefGoogle Scholar
  33. 33.
    Cremer T et al (2000) Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. Crit Rev Eukaryot Gene Expr 10(2):179–212PubMedCrossRefGoogle Scholar
  34. 34.
    Mirny LA (2011) The fractal globule as a model of chromatin architecture in the cell. Chromosome Res 19(1):37–51PubMedCrossRefGoogle Scholar
  35. 35.
    Rouquette J, Cremer C, Cremer T, Fakan S (2010) Functional nuclear architecture studied by microscopy: present and future. Int Rev Cell Mol Biol 282:1–90PubMedCrossRefGoogle Scholar
  36. 36.
    Mor A et al (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12(6):543–552PubMedCrossRefGoogle Scholar
  37. 37.
    Albiez H et al (2006) Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chromosome Res 14(7):707–733PubMedCrossRefGoogle Scholar
  38. 38.
    Albiez H (2007) Manipulation of global chromatin architecture in the human cell nucleus and critical assessment of current model views. Ludwig-Maximilians-University, Munich, DissertationGoogle Scholar
  39. 39.
    Bornfleth H, Edelmann P, Zink D, Cremer T, Cremer C (1999) Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy. Biophys J 77(5):2871–2886PubMedCrossRefGoogle Scholar
  40. 40.
    Strickfaden H, Zunhammer A, van Koningsbruggen S, Kohler D, Cremer T (2010) 4D chromatin dynamics in cycling cells: Theodor Boveri’s hypotheses revisited. Nucleus 1(3):284–297PubMedCrossRefGoogle Scholar
  41. 41.
    van Steensel B, Dekker J (2010) Genomics tools for unraveling chromosome architecture. Nat Biotechnol 28(10):1089–1095PubMedCrossRefGoogle Scholar
  42. 42.
    Diaz G, Isola R, Falchi AM, Diana A (1999) CO2-enriched atmosphere on the microscope stage. Biotechniques 27:292–294PubMedGoogle Scholar
  43. 43.
    Spierenburg GT, Oerlemans FT, van Laarhoven JP, de Bruyn CH (1984) Phototoxicity of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered culture media for human leukemic cell lines. Cancer Res 44(5):2253–2254PubMedGoogle Scholar
  44. 44.
    Hübner B, Strickfaden H, Müller S, Cremer M, Cremer T (2009) Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure. Eur Biophys J 38(6):729–747PubMedCrossRefGoogle Scholar
  45. 45.
    Markaki Y, Smeets D, Cremer M, Schermelleh L (2013) Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy. Methods Mol Biol 950:43–64PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Barbara Hübner
    • 1
  • Thomas Cremer
    • 1
  • Jürgen Neumann
    • 2
  1. 1.Department Biology II, Anthropology and Human Genetics, BiocenterLudwig-Maximilians-University (LMU)MartinsriedGermany
  2. 2.Department Biology II, Human Biology and BioImaging, BiocenterLudwig-Maximilians-University (LMU)MartinsriedGermany

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