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

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Imaging Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1042))

Abstract

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.

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Abbreviations

1 Mbp CDs:

Megabase-sized chromatin domains

3D:

Three-dimensional

3D-SIM:

Three-dimensional structured illumination microscopy

4D:

Four-dimensional

CC:

Chromatin compartment

CLSM:

Confocal laser scanning microscopy

CT–IC:

Chromosome territory–interchromatin compartment

EM:

Electron microscopy

FCS:

Fetal calf serum

FIB:

Focused ion beam

FISH:

Fluorescence in situ hybridization

FRAP:

Fluorescence recovery after photobleaching

GFP:

Green fluorescent protein

H2B-mRFP:

Histone 2B tagged with red fluorescent protein

H3K4me3:

Histone 3 tri-methylated at lysine 4

H3K9me3:

Histone 3 tri-methylated at lysine 9

HCC:

Hypercondensed chromatin

IC:

Interchromatin compartment

OTF:

Optical transfer function

PBS:

Phosphate buffered saline

PBST:

1× PBS with 0.02 % Tween

PFA:

Paraformaldehyde

PR:

Perichromatin region

PS:

Penicillin/streptomycin

PSF:

Point-spread function

RNPs:

Ribonucleoproteins

ROI:

Region of interest

SDLSM:

Spinning disk laser scanning microscopy

SEM:

Scanning electron microscopy

SIM:

Structured illumination microscopy

TEM:

Transmission electron microscopy

WF:

Wide field

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Acknowledgements

This work was supported by grants to Thomas Cremer (DFG grant SFB684, CR-59/29-2).

We are indebted to Stanislav Fakan and Jacques Rouquette for introducing us into the techniques of osmium ammine staining for DNA and TEM and to Yolanda Markaki for helping with establishing immunofluorescence procedures for 3D-SIM. We thank our colleagues Dirk Eick for providing the RNA polymerase II antibodies, Otto Berninghausen for technical support with TEM, and Heinrich Leonhardt for continued support of our studies.

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Authors and Affiliations

  1. Department Biology II, Anthropology and Human Genetics, Biocenter, Ludwig-Maximilians-University (LMU), Martinsried, Germany

    Barbara Hübner & Thomas Cremer

  2. Department Biology II, Human Biology and BioImaging, Biocenter, Ludwig-Maximilians-University (LMU), Martinsried, Germany

    Jürgen Neumann

Authors
  1. Barbara Hübner
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  2. Thomas Cremer
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Editor information

Editors and Affiliations

  1. Bar-Ilan University Inst. of Nanotech & Advanced Materials, Ramat Gan, Israel

    Yaron Shav-Tal

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Hübner, B., Cremer, T., Neumann, J. (2013). Correlative Microscopy of Individual Cells: Sequential Application of Microscopic Systems with Increasing Resolution to Study the Nuclear Landscape. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 1042. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-526-2_21

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  • DOI: https://doi.org/10.1007/978-1-62703-526-2_21

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-525-5

  • Online ISBN: 978-1-62703-526-2

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