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We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.
The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.
Light microscopy and, more particularly, fluorescence microscopy are robust and versatile techniques commonly used in biological sciences. They give access to the precise localization of various biomolecules like proteins or RNA through their specific fluorescent labeling. The last decade has been characterized by rapid advances in microscopy and imaging technologies as evidenced by the 2014 Nobel Prize in Chemistry awarding Eric Betzig, Stefan W. Hell and William E. Moerner for the development of super-resolved fluorescence microscopy (SRFM)1. SFRM bypasses the diffraction limit of traditional optical microscopy to bring it into the nanodimens....
NOTE: User-friendly tutorials are available on Icy’s website http://icy.bioimageanalysis.org.
1. Download Icy and the Substructure Analyzer protocol
All the described analyses have been performed on a standard laptop (64-bit, quad-core processor at 2.80 GHz with 16 GB random-access memory (RAM)) working with the 64-bit version of Java. Random-access memory is an important parameter to consider, depending on the amount and the resolution of images to analyze. Using the 32-bit version of Java limits the memory to about 1300 MB, which could be unsuitable for big data analysis, whereas the 64-bit version allows increasing the memory allocated to Icy.
An increasing number of free software tools are available for the analysis of fluorescence cell images. Users must correctly choose the adequate software according to the complexity of their problematic, to their knowledge in image processing, and to the time they want to spend in their analysis. Icy, CellProfiler, or ImageJ/Fiji are powerful tools combining both usability and functionality3. Icy is a stand-alone tool that presents a clear graphical user interface (GUI), and notably its “Pro.......
The authors have nothing to disclose.
G.H. was supported by a graduate fellowship from the Ministère Délégué à la Recherche et aux Technologies. L.H. was supported by a graduate fellowship from the Institut de Cancérologie de Lorraine (ICL), whereas Q.T. was supported by a public grant overseen by the French National Research Agency (ANR) as part of the second “Investissements d’Avenir” program FIGHT-HF (reference: ANR-15-RHU4570004). This work was funded by CNRS and Université de Lorraine (UMR 7365).
....Name | Company | Catalog Number | Comments |
16% Formaldehyde solution (w/v) methanol free | Thermo Fisher Scientific | 28908 | to fix the cells |
Alexa Fluor 488 of goat anti-rabbit | Thermo Fisher Scientific | A-11008 | fluorescent secondary antibody |
Alexa Fluor 555 of goat anti-mouse | Thermo Fisher Scientific | A-21425 | fluorescent secondary antibody |
Alexa Fluor 555 Phalloidin | Thermo Fisher Scientific | A34055 | fluorescent secondary antibody |
Bovine serum albumin standard (BSA) | euromedex | 04-100-812-E | |
DMEM | Sigma-Aldrich | D5796-500ml | cell culture medium |
Duolink In Situ Mounting Medium with DAPI | Sigma-Aldrich | DUO82040-5ML | mounting medium |
Human: HeLa S3 cells | IGBMC, Strasbourg, France | cell line used to perform the experiments | |
Hydrogen peroxide solution 30% (H2O2) | Sigma-Aldrich | H1009-100ml | used as a stressing agent |
Lipofectamine 2000 Reagent | Thermo Fisher Scientific | 11668-019 | transfection reagent |
Mouse monoclonal anti-coilin | abcam | ab11822 | Coilin-specific antibody |
Nikon Optiphot-2 fluorescence microscope | Nikon | epifluoresecence microscope | |
Opti-MEM I Reduced Serum Medium | Thermo Fisher Scientific | 31985062 | transfection medium |
PBS pH 7.4 (10x) | gibco | 70011-036 | to wash the cells |
Rabbit polyclonal anti-53BP1 | Thermo Fisher Scientific | PA1-16565 | 53BP1-specific antibody |
Rabbit polyclonal anti-EDC4 | Sigma-Aldrich | SAB4200114 | EDC4-specific antibody |
Triton X-100 | Roth | 6683 | to permeabilize the cells |
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