Protocol

Imaging Gene Expression

Volume 1042 of the series Methods in Molecular Biology pp 299-336

Date:

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

  • Barbara HübnerAffiliated withDepartment Biology II, Anthropology and Human Genetics, Biocenter, Ludwig-Maximilians-University (LMU)
  • , Thomas CremerAffiliated withDepartment Biology II, Anthropology and Human Genetics, Biocenter, Ludwig-Maximilians-University (LMU)
  • , Jürgen NeumannAffiliated withDepartment Biology II, Human Biology and BioImaging, Biocenter, Ludwig-Maximilians-University (LMU)

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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.

Key words

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