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Nat Protoc. 2014 Mar;9(3):586-96. doi: 10.1038/nprot.2014.037. Epub 2014 Feb 13.

Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

Author information

1
1] Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, Villigen PSI, Switzerland. [2] MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
2
1] MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. [2].
3
MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
4
Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, Villigen PSI, Switzerland.
5
ETH Zurich, Institute of Molecular Health Sciences, Zürich, Switzerland.
6
Center for Microscopy and Image Analysis, University of Zurich, Zürich, Switzerland.

Abstract

Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires <1 min and <5 min for 2D and 3D images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

PMID:
24525752
DOI:
10.1038/nprot.2014.037
[Indexed for MEDLINE]

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