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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:44601*  Cite 
Description

Shown is a mitotic PtK2 cell at metaphase immunostained for microtubules (red) and kinetochores (green) with DNA stained blue. The image was obtained using structured illumination microscopy (SIM, Deltavision OMX system) which provides 'super-resolution' beyond the diffraction limit set by the wavelength of the illuminating light. The micrograph was the 2012 winner in the high- and super-resolution microscopy category of the GE Healthcare Life Sciences Imaging Contest, and featured in the NIGMS Biomedical Beat, the monthly digest of notable NIGMS-sponsored research.

Technical Details

PtK2 cells were grown on poly-L-lysine-coated cover glasses, treated briefly with an extraction buffer containing CaCl2 and Triton X-100 to remove unpolymerized tubulin, followed by fixation for 20 min with 1% glutaraldehyde in extraction buffer. After quenching in sodium borohydride, cells were immunostained for microtubules (primary antibody mouse DM1-alpha, secondary antibody donkey anti-mouse [-Xrat] Dylight 594, red), kinetochores (primary antibody rabbit anti-CenpE, secondary antibody donkey anti-rabbit-Alexa 488, green), and chromosomes counterstained blue with Hoechst dye. Images were captured and processed using a Deltavision OMX 3D-SIM Super-Resolution microscopy system in which structured illumination provides resolution that exceeds the diffraction limit. Through-focus Z-stacks of images were obtained with a 0.125 micrometer step size and a 100x 1.4 NA UNIPLANAPO objective lens, and the z-slices subsequently processed and projected down the z-axis.

Biological Sources
NCBI Organism Classification
Hypsiprymnodon moschatus
Cell Type
kidney epithelial cell
Cell Line
PtK2
Cellular Component
mitotic spindle
nuclear chromosome
kinetochore
Biological Context
Biological Process
mitosis
mitotic metaphase
Attribution
Names
Jane Stout, Indiana University
Claire Walczak, Indiana University
Link
NIGMS Biomedical Beat
GE Healthcare Imaging Competition
Citation
Digital Object Identifier (DOI)
doi:10.7295/W9CIL44601
Archival Resource Key (ARK)
ark:/b7295/w9cil44601
Imaging
Image Type
recorded image
Image Mode
fluorescence microscopy
structured illumination microscopy (SIM)
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of epitope
Visualization Methods
Hoechst 33342
Alexa Fluor 488
DyLight 594
Data Qualifiers
processed data
suitable for spatial measurements
Sample Preparation
Methods
glutaraldehyde fixed tissue
Relation To Intact Cell
whole mounted tissue
Dimensions
Spatial Axis Image Size Pixel Size
X 1500px ——
Y 1500px ——