Method Article

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

DOI:

10.3791/60990

⸱

July 15th, 2020

 ,  ,  ,  ,  ,  , 
1Université de Lorraine, CNRS, IMoPA F54000 Nancy France, 2Institut Pasteur, BioImage Analysis Unit, CNRS UMR 3691, Paris, France.

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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....

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Protocol

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NOTE: User-friendly tutorials are available on Icy’s website http://icy.bioimageanalysis.org.

1. Download Icy and the Substructure Analyzer protocol

  1. Download Icy from the Icy website (http://icy.bioimageanalysis.org/download) and download the Substructure Analyzer protocol: http://icy.bioimageanalysis.org/protocols?sort=latest.
    NOTE: If using a 64-bit OS, be sure to use the 64-bit version of Java. This version allows for increasing the memory allocated to Icy (Preferences | General | Ma....

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Results

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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.

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Discussion

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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.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
16% Formaldehyde solution (w/v) methanol freeThermo Fisher Scientific28908to fix the cells
Alexa Fluor 488 of goat anti-rabbitThermo Fisher ScientificA-11008fluorescent secondary antibody
Alexa Fluor 555 of goat anti-mouseThermo Fisher ScientificA-21425fluorescent secondary antibody
Alexa Fluor 555 PhalloidinThermo Fisher ScientificA34055fluorescent secondary antibody
Bovine serum albumin standard (BSA)euromedex04-100-812-E
DMEMSigma-AldrichD5796-500mlcell culture medium
Duolink In Situ Mounting Medium with DAPISigma-AldrichDUO82040-5MLmounting medium
Human: HeLa S3 cellsIGBMC, Strasbourg, Francecell line used to perform the experiments
Hydrogen peroxide solution 30% (H2O2)Sigma-AldrichH1009-100mlused as a stressing agent
Lipofectamine 2000 ReagentThermo Fisher Scientific11668-019transfection reagent
Mouse monoclonal anti-coilinabcamab11822Coilin-specific antibody
Nikon Optiphot-2 fluorescence microscopeNikonepifluoresecence microscope
Opti-MEM I Reduced Serum MediumThermo Fisher Scientific31985062transfection medium
PBS pH 7.4 (10x)gibco70011-036to wash the cells
Rabbit polyclonal anti-53BP1Thermo Fisher ScientificPA1-1656553BP1-specific antibody
Rabbit polyclonal anti-EDC4Sigma-AldrichSAB4200114EDC4-specific antibody
Triton X-100Roth6683to permeabilize the cells

References

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  1. Möckl, L., Lamb, D. C., Bräuchle, C. Super-resolved fluorescence microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William E. Moerner. Angewandte Chemie. 53 (51), 13972-13977 (2014).
  2. Meijering, E., Carpenter, A. E., Peng, H., Hamprecht, F. A., Olivo-Marin, J. -C.

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Tags

Substructure AnalyzerFluorescence MicroscopyImage SegmentationBioimage AnalysisCell Body DetectionSignal QuantificationIcy PlatformImageJ IntegrationNuclear TranslocationCajal Bodies

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