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. 2017 Feb 20;7(4):e2145.
doi: 10.21769/BioProtoc.2145.

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Vincent Bayle  1 , Matthieu Pierre Platre  1 , Yvon Jaillais  1
Affiliations

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Vincent Bayle et al. Bio Protoc. .

Abstract

In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species.

Keywords: Arabidopsis; Compartment; Confocal analysis; Endocytosis; Image segmentation; Root; Spot detection.

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Figures

Video 1.
Video 1.. Procedure to install the Sice Spotdetector Macro and related Plugins
Figure 1.
Figure 1.. Snapshot from FIJI program showing manual measurement of individual spot-like structures using plot-profile and straight-line tools (step C3).
A. FIJI main toolbar; B. Test image window; C. Enlargement of the dashed-square in (B), showing the red line that is drawn across several compartments using the straight-line tool of ImageJ. D. Fluorescence plot along the red line. Compartments are identified as peaks and are measured by drawing lines at their base. E. Peak width measurements.
Video 2.
Video 2.. Procedure to estimate the size of the compartments of interest
Figure 2.
Figure 2.. Snapshot from ImageJ program showing the size of different intracellular compartments as captured by spinning disk microscopy.
A. FIJI main toolbar; B. Golgi apparatus, marker line is Wave18 (W18) ( Geldner et al., 2009 ). C. Post-Golgi endosomal compartments, marker line is Wave25 (W25) ( Geldner et al., 2009 ). D. Late endosomal compartments, marker line is Wave7 (W7) ( Geldner et al., 2009 ). E. Wortmannin compartments. Late endosomes (W7) fuse into the so-called ‘Wortmannin compartment’ following treatment with the PI3Kinase/PI4Kinase inhibitor Wortmannin (Wm, 30 µM for 60 min) ( Jaillais et al., 2006 ; Simon et al., 2014 ; Simon et al., 2016 ). F. BFA compartments. Endosomes labeled by the endocytic tracer FM4-64 aggregates into the so-called ‘BFA compartment’ following treatment with the fungal toxin BrefeldinA (BFA, 50 µM for 60 min) ( Geldner et al., 2003 ; Geldner et al., 2009 ). G. Summary table of peak width measurements for the marker shown in A to F.
Video 3.
Video 3.. Procedure to find the best parameters to segment intracellular particles with the SiCE spot detector macro
Figure 3.
Figure 3.. Snapshot from ImageJ program showing selection of parameters step.
A. FIJI main toolbar; B. Test image window; C. Select Parameters selection windows of the SICE spot detector.
Figure 4.
Figure 4.. Snapshot from ImageJ program showing the results for single image analysis.
A. FIJI main toolbar; B. Test image window. Region of Interest is represented as a red line. C. Result of spot detection using the following parameters: MinSize 0.5 MaxSize 99 circMin 0.5 circMax 1 Sigma 3 Filtering Method Difference of Gaussian Threshold Triangle; D. Result of spot detection using the following parameters: MinSize 1 MaxSize 99 circMin 0.5 circMax 1 Sigma 3 Filtering Method Difference of Gaussian Threshold Triangle; D. ‘Result summary’ table showing the quantification of the number of spot in the different condition tested, using the parameters defined as in C for line 1 and as in D for line 2.
See this image and copyright information in PMC

References

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