Computational protocol: Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

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Protocol publication

[…] Indirect quantification of fluorescence levels, maximum intensity projections and kymograph construction were performed using ImageJ software []. Kymographs were constructed with our in-house developed plug-in package for ImageJ software, which is freely available from our website . [...] To assess the cell plate structures, we first obtained cross-sectional images from 70 serial optical sections taken at 0.5 μm intervals of each cell with view orthogonal planes function in MetaMorph image-analysis software (Universal Imaging Co., Downingtown, Panama). Subsequently, we obtained the binary images from the cross-sectional images by intensity thresholding using ImageJ software as shown in Figure . To improve the signal to noise ratio, a Gaussian filter was applied to the cross-sectional images before binarization. Cell plate area and perimeter were calculated from the binary images with ImageJ software. Complexity, a secondary morphological parameter, was calculated by P2/4πA, where P is the perimeter and A is the area, according to Tanaka et al. (2007) []. [...] To estimate the velocity of endosomal movement, we performed time-sequential observations at 1-sec intervals for 30 sec by spinning-disc confocal laser microscopy. From the time-sequential images, we measured the distance and time traveled by endosomes using the ImageJ plug-in MTrack2, which was developed for multiple particle detection and tracking, and which is freely available from the website . Subsequently, endosomal velocity was calculated by dividing the distance traveled by the traveling time. […]

Pipeline specifications

Software tools ImageJ, MetaMorph, Mtrack2
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Nicotiana tabacum, Rattus norvegicus
Diseases Abnormalities, Drug-Induced