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

Summary

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

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

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....

Protocol

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 | Gene....

Results

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.

Discussion

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.......

Materials

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