Computational protocol: Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants

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Protocol publication

[…] Seven-day-old seedlings were imaged using a stereo microscope (Leica M165 FC). Root lengths were measured using Image J ( Statistical analysis was performed using a two-tailed t-test to assess differences between the mean values from the control and the propargylcholine-grown roots. [...] Co-localization of the fluorescence signals from fluorescein azide or Alexa Fluor 594 azide marking propargylcholine (pp-Cho) and various organelle markers (DAPI for nuclei; GFP or YFP for mitochondria, ER, Golgi or tonoplast), cell wall stain propidium iodide and chlorophyll fluorescence were quantified for the two separate channels for four regions of interest (ROI) from each confocal image using JACoP (Just Another Co-localization Plugin) on the public domain ImageJ software []. Pearson correlation coefficient (PCC) values were calculated to estimate the degree of co-localization. PCC values range from + 1 to − 1, with + 1 representing complete positive correlation and − 1 representing negative correlation and 0 value representing no correlation. […]

Pipeline specifications

Software tools ImageJ, JACoP
Application Microscopic phenotype analysis
Organisms Arabidopsis thaliana
Chemicals Choline, Phosphatidylcholines, Fluorescein