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

  • Barbara Hübner
  • Thomas Cremer
  • Jürgen Neumann
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

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

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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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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