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Nat Protoc. 2017 May;12(5):1011-1028. doi: 10.1038/nprot.2017.020. Epub 2017 Apr 13.

Quantitative 3D structured illumination microscopy of nuclear structures.

Author information

1
Center for Integrated Protein Science (CIPSM) and Center for Advanced Light Microscopy (CALM), Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
2
Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, Oxford, UK.
3
Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Kobe, Japan.
4
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

Abstract

3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.

PMID:
28406495
DOI:
10.1038/nprot.2017.020
[Indexed for MEDLINE]

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