Quantitative 3D structured illumination microscopy of nuclear structures

Kraus, Felix; Miron, Ezequiel; Demmerle, Justin; Chitiashvili, Tsotne; Budco, Alexei; Alle, Quentin; Matsuda, Atsushi; Leonhardt, Heinrich; Schermelleh, Lothar und Markaki, Yolanda (2017): Quantitative 3D structured illumination microscopy of nuclear structures. In: Nature Protocols, Bd. 12, Nr. 5: S. 1011-1028

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie > Department Biologie II
• Fakultätsübergreifende Einrichtungen: Center for Integrated Protein Science Munich (CIPSM)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 1754-2189
• Sprache: Englisch