title: Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.
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.
authors: 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.
authors: Aurélien
authors: A
authors: Rizk
authors: 1] MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. [2].
authors: Grégory
authors: G
authors: Paul
authors: MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors: Pietro
authors: P
authors: Incardona
authors: Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, Villigen PSI, Switzerland.
authors: Milica
authors: M
authors: Bugarski
authors: Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, Villigen PSI, Switzerland.
authors: Maysam
authors: M
authors: Mansouri
authors: ETH Zurich, Institute of Molecular Health Sciences, Zürich, Switzerland.
authors: Axel
authors: 0000000229300950
authors: A
authors: Niemann
authors: Center for Microscopy and Image Analysis, University of Zurich, Zürich, Switzerland.
authors: Urs
authors: U
authors: Ziegler
authors: Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, Villigen PSI, Switzerland.
authors: Philipp
authors: P
authors: Berger
authors: MOSAIC Group, Center of Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors: Ivo F
authors: IF
authors: Sbalzarini
mesh_terms: D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012984:Software; D013347:Subcellular Fractions / Q000648:ultrastructure*