TY - JOUR
T1 - Quantitative 3D structured illumination microscopy of nuclear structures
AU - Kraus, Felix
AU - Miron, Ezequiel
AU - Demmerle, Justin
AU - Chitiashvili, Tsotne
AU - Budco, Alexei
AU - Alle, Quentin
AU - Matsuda, Atsushi
AU - Leonhardt, Heinrich
AU - Schermelleh, Lothar
AU - Markaki, Yolanda
PY - 2017/4/13
Y1 - 2017/4/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85019731688&partnerID=8YFLogxK
U2 - 10.1038/nprot.2017.020
DO - 10.1038/nprot.2017.020
M3 - Article
C2 - 28406495
AN - SCOPUS:85019731688
SN - 1754-2189
VL - 12
SP - 1011
EP - 1028
JO - Nature Protocols
JF - Nature Protocols
IS - 5
ER -