Build kymographs tracing a line ROI across the mitochondria that was illuminated with Blue Light.

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.

Normalize the fluorescence intensities to the min and max values using the following formula:

Normalized f[t] = f[t]-f_min[t]

                              ————————

                            f<sub>max</sub>[t]−f<sub>min</sub>[t]

where f[t] = F_mito[t] − F_bkgd[t].

Analyze the cells coexpressing VPS13D^EGFP and mitoBFP individually. Store the each cell outline as an ROI in the ROI Manger of Fiji.

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.

Use an automated macro designed in Fiji to measure VPS13D^EGFP signal overlapping with mitochondria and that localized in the cytosol surrounding them.

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 the

saving 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 set

the saving directory

count = roiManager("Count");//save the information from ROI manager into the term "count"

for (i = 0; i < count; 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 in

batch

        saveAs("Tiff", dir_saving + "ROIslice"+i+name); //save the selected ROIs in the ROI manager in

batch

        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, click

OK. If different, click Cancel and make changes.\n");

                       Dialog.addMessage("Channel 1 is VPS13D channel. Channel 2 is Mitochondrial

channel.");

                        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=3

mask=None fast_(less_accurate)");//clearify the outline of mitochondria

            run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3

mask=None fast_(less_accurate)");//clearify the outline of mitochondria

            run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3

mask=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 mitomask

onto 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 to

mitochondrial.csv");

            close()



            }



            Dialog.create("reminder");



                        Dialog.addMessage("Mission accomplished.Congratulations!");



                        Dialog.show();

}