Quantitative 3D structured illumination microscopy of nuclear structures

Felix Kraus, Ezequiel Miron, Justin Demmerle, Tsotne Chitiashvili, Alexei Budco, Quentin Alle, Atsushi Matsuda, Heinrich Leonhardt, Lothar Schermelleh, Yolanda Markaki

Research output: Contribution to journalArticleOtherpeer-review

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1011-1028
Number of pages18
JournalNature Protocols
Volume12
Issue number5
DOIs
Publication statusPublished - 13 Apr 2017

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