Computational protocol: Optogenetic protein clustering through fluorescent protein tagging and extension of CRY2

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Protocol publication

[…] Images were analyzed with Nikon imaging software (NIS-elements AR 64-bit version 3.21; Laboratory Imaging), MetaMorph software (version 7.8.1.0; MDS Analytical Technologies) or ImageJ software (version 1.50b; U.S. National Institutes of Health; http://imagej.nih.gov/ij/). For quantification of cluster formation, clusters were defined as discrete puncta of fluorescence with criteria of fluorescence intensity (1,500–4,095 arbitrary units) and equivalent diameter (>0.2 μm). The percentage of clustered cells was calculated by dividing the number of cells that formed clusters upon blue light stimulation by the total population of transfected cells. The cluster ratio in cells was quantified using “Automated Measurement” and “ROI Statistics” tools in Nikon imaging software. The total cluster intensity (I C) was divided by the total fluorescence intensity (I W) of the whole cell. Cluster size distribution was determined by classifying different cluster areas using the “Annotated Measurement Results” in Nikon imaging software. Co-localization between two molecules with different fluorescent colors was quantified with Pearson’s correlation coefficient by using the “Co-localization” tool in Nikon imaging software. A “Kymograph” tool in MetaMorph software was used to draw kymographs. Statistical significance was evaluated using a two-tailed Student’s t-test. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Arabidopsis thaliana