TITLE: Protocol for Image processing and analysis of VPS13D recruitment to mitochondria
AUTHORS: Marianna Leonzino, Andrés Guillén-Samander, Ni Tang, Pietro De Camilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details the image processing and analysis of VPS13D recruitment to mitochondria as it was performed in </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1083/jcb.202010004</span></a><span>. The first part details the analysis of acute optogenetic recruitment, and the second part of basal recruitment under different conditions.</span></div></div>

[STEPS]
?. [For the analysis of optogenetic experiments:]
Build kymographs tracing a line ROI across the mitochondria that was illuminated with Blue Light.
?. [For the analysis of optogenetic experiments:]
Measure an intensity profile by tracing a line ROI across the center of the mitochondrial signal on the kymograph. Take a background profile using the same line ROI outside the illumination area.
?. [For the analysis of optogenetic experiments:]
Normalize the fluorescence intensities to the min and max values using the following formula: Normalized f[t] = f[t]-fmin[t]                                  ————————                                fmax[t]−fmin[t]where f[t] = Fmito[t] − Fbkgd[t].
?. [For the analysis of VPS13D mitochondrial recruitment:]
Analyze the cells coexpressing VPS13D^EGFP and mitoBFP individually. Store the each cell outline as an ROI in the ROI Manger of Fiji.
?. [For the analysis of VPS13D mitochondrial recruitment:]
Apply the “Gaussian Blur” tool and “Enhance Local Contrast” tool to the MitoBFP channel to average the brightness of the whole image and to clarify the shape and margin of mitochondria.
?. [For the analysis of VPS13D mitochondrial recruitment:]
Use an automated macro designed in Fiji to measure VPS13D^EGFP signal overlapping with mitochondria and that localized in the cytosol surrounding them.
?. [For the analysis of VPS13D mitochondrial recruitment:]
Calculate the ratio between the intensity of VPS13D^EGFP on mitochondria and the intensity of VPS13D^EGFP in the cytosol surrounding mitochondria for each cell and plot a graph indicating the enrichment of VPS13D^EGFP on mitochondria.The macro (which can be adapted to analyse recruitment of any given protein to mitochondria) is included below:macro "ROI in batches auto measure VPS13 binding with mitochondrial " {Dialog.create("reminder");//create an interface                                Dialog.addMessage("choose saving path");//an interactive interface for user to set thesaving directory                                Dialog.show();//an interactive interface for user to set the saving directory                dir_saving = getDirectory("Choose a Directory to save");//an interactive interface for user to setthe saving directorycount = roiManager("Count");//save the information from ROI manager into the term "count"for (i = 0; i             name = getTitle;            selectWindow(name);            roiManager("Select", i);//save the selected ROIs in the ROI manager in batch            Stack.getPosition(channel, slice, frame);//save the selected ROIs in the ROI manager in batch            run("Duplicate...", "duplicate slices=" + slice);//save the selected ROIs in the ROI manager inbatch            saveAs("Tiff", dir_saving + "ROIslice"+i+name); //save the selected ROIs in the ROI manager inbatch            name = replace(name, ".tif", "");//motify the name of images            C1 = "C1-ROIslice"+i+name+".tif";//motify the name of images            C2 = "C2-ROIslice"+i+name+".tif";//motify the name of images            run("Split Channels");//split the integrated image into single channel            Dialog.create("Content of the channel");//create an interface                          Dialog.addMessage("Make sure the content of the channels. If same as described, clickOK. If different, click Cancel and make changes.\n");                           Dialog.addMessage("Channel 1 is VPS13D channel. Channel 2 is Mitochondrialchannel.");                            Dialog.show();//make sure the content of each channels                selectWindow(C1);//choose the cytosol slice                run("Duplicate...", " ");//duplicate a seperate image to process                run("Brightness/Contrast...");//preprocess the VPS13D signal in the cytosol area//measure the intensity of VPS13D signal in mitochondria area                selectWindow(C2);//choose the mitochondria slice                run("Duplicate...", " ");//duplicate a seperate image to process                run("Gaussian Blur...", "sigma=2 slice");//balance the brightness of image                run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3mask=*None* fast_(less_accurate)");//clearify the outline of mitochondria                run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3mask=*None* fast_(less_accurate)");//clearify the outline of mitochondria                run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3mask=*None* fast_(less_accurate)");//clearify the outline of mitochondria                run("Threshold...");//choose the mitochondria area                run("Convert to Mask");//generate an accurate mitomask                saveAs("Tiff", dir_saving + "ROI"+i+" mitomask "+name);//generate an accurate mitomask                imageCalculator("AND create", mitomask, C1);//map C1 onto the mitomask                setThreshold(1, 255);//choose the calculated area                saveAs("Tiff", dir_saving + "ROI"+i+" result of mitochondria intensity "+name);                mitointensity=getTitle;                run("Measure");//measure the intensity of VPS13D signal in mitochondria area//measure the VPS13D intensity in the cytosol area                run("Dilate");                run("Dilate");                run("Dilate");//duplicate three times to eliminate the interference from mitochondria signal                run("Convert to Mask");                run("Invert");//invert the enlarged mitomask                saveAs("Tiff", dir_saving + "ROI"+i+" mitomask x3 "+name);//generate the enlarged inverted                mitomask                mitomask3 = getTitle;                imageCalculator("AND create", mitomask, mitomask);//map the enlarged inverted mitomaskonto the original mitomask to get the enlarged outline of mitochondria in the cytosol area                saveAs("Tiff", dir_saving + "ROI"+i+" cytomask "+name);//generate outline profile of mitomask,which refers to mask of cytosol area                cytomask = getTitle;                imageCalculator("AND create", cytomask, C1);//map the cytomask onto cytosol                setThreshold(1,255);                saveAs("Tiff", dir_saving + "ROI"+i+" result of cytosolic intensity "+name);                cytointensity=getTitle;                run("Measure");//measure the VPS13D intensity in the cytosol area                saveAs("Results", dir_saving + "ROI"+i+" measurement results of VPS13 binding tomitochondrial.csv");                close()                }                Dialog.create("reminder");                            Dialog.addMessage("Mission accomplished.Congratulations!");                            Dialog.show();}