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Quantitative 3D structured illumination microscopy of nuclear structures.

Felix Kraus1, Ezequiel Miron2, Justin Demmerle2

  • 1Center for Integrated Protein Science (CIPSM) and Center for Advanced Light Microscopy (CALM), Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.

Nature Protocols
|April 14, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a validated protocol for 3D structured illumination microscopy (3D-SIM) to analyze nuclear structures and epigenetic marks in mammalian cells. The method enables high-resolution, quantitative imaging and analysis within one week.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Epigenetics

Background:

  • 3D structured illumination microscopy (3D-SIM) is crucial for multicolor volumetric super-resolution imaging.
  • Analyzing the complex nuclear environment with 3D-SIM presents significant technical challenges.
  • Existing protocols lack comprehensive solutions for nuclear structure and epigenetic mark analysis using 3D-SIM.

Purpose of the Study:

  • To provide a validated protocol for 3D-SIM in cultured mammalian cells, focusing on nuclear structures and epigenetic modifications.
  • To establish practical guidelines and open-source solutions for high-quality, comparable data generation.
  • To enable quantitative analysis of 3D nuclear organization and epigenetic marks at the nanometer scale.

Main Methods:

  • Development of a sample preparation protocol including immunostaining and replication labeling.
  • Image acquisition and registration techniques optimized for the nuclear environment.
  • Application of image segmentation, centroid mapping, and nearest-neighbor analyses using open-source software (ImageJ/Fiji, TANGO plugin).

Main Results:

  • Successful application of 3D-SIM to label and analyze nuclear structures and epigenetic marks.
  • Generation of 3D nuclear maps with nanometer-scale resolution and normalization to fluorescence standards.
  • Demonstration of quantitative analysis of nuclear substructures and epigenetic modifications.

Conclusions:

  • The protocol effectively addresses the challenges of applying 3D-SIM to the nuclear environment.
  • The open-source solutions facilitate routine, high-quality data generation across different model systems.
  • This work enables precise, quantitative investigation of 3D nuclear organization and its epigenetic regulation.