Here, you find ImageJ Macros to automatize the analysis of membrane contact sites (MCSs) between mitochondria and endosomes/lysosomes (endo-lysosomes) based on two-color confocal fluorescence microscopy data. Macros are provided for analysis in two and three dimensions based on (x,y) and (x,y,z) image data, respectively.
First, you identify the x,y-position of all endo-lysosomes from a fluorescence image of suitably labeled cells. To identiy endo-lysosomes various markers can be used (e.g. fluorescent dextran or immunos-taining using antibodies against Lamp1). You open your image within ImageJ/Fiji and run /Process/FindMaxima to find an appropriate value for the Prominence using 'Preview Point selection', but without applying it. You download and run the Macro 'Find_spot_maxima_f.ijm' using the found prominence value, giving you a list of ROIs assembled in one ROI named 'Points' in the ROI Manager of ImageJ.
Second, you download the Macro 'Skeletonize_spotDistance_EDT.ijm' and run it on the corresponding image of labeled mitochondria. For labeling MitoTracker or immunostaining (e.g. against VDAC or TOM20) can be used. Provide first the pixel size of your images, which you can find from the TIFF header under /Image/Properties in ImageJ. Click 'OK' and choose a threshold to identify the mitochondria based on their intensity. Click 'OK, and the Macro will a binarized skeleton of the mitochondria and from that an Euclidian distance transform (a new image called 'EDT'). It will map the positions of the identified endo-lysosomes onto the EDT and output the intensity value into the Results table. These intensity values can be found as 'Mean intensity' in the Results table (if this was chosen under /Analyze/Set Measurements, and they are the distance of each endo.lysosome peak position to the next mitochondrion in µm.
For 3D analysis, you need confocal z-stacks of doubly-labeled cells and have to have the 3D ImageJ suite deveoped by Drs. Thomas Boudier and Jean Ollion installed (available at https://imagej.net/3D_ImageJ_Suite). Make sure, that you have defined the pixel dimensions in x, y and z according to your micorscopy settings. Run the Macro 'Run_3D_analysis.ijm' on the z-stack of your endo-lysosome marker (evtl. adjust threshold values). This takes a few seconds depending on the processing power of your computer and your image sizes. Duplictae those frames of the generated centroid map, which contains pixel values larger than zero, in a new stack. Run the Macro 'Find_spot_maxima_3D_f.ijm' on this stack. Save all the generated image stacks. Open the corresponding mitochondria confocal z-stack, generate a binary image of that using /Image/Adjust/Threshold in ImageJ and run the 3D Distance map plugin from the 3D ImageJ analysis suite on that image stack. Use 'Invert' as option. The generated EDT map will account for the asymmetric dimensions of voxels. Select the frames of this EDT stack corresponding to those of the centroid map by duplicating them into a new stack. Run the Macro 'Spot_Distance_EDT_3D.ijm'. It will give you the distance of each endo-lysosome centroid from the nearest mitochondrium as intensity value in the Results table.
Further details of the image analysis workflow, an alternative approach for 3D analysis and results of it can be found in the publication:
Dupont Juhl, A., Heegaard, C.W., Werner, S., Schneider, G., Krishnan, K., Covey, D.F. and Wüstner, D. 2021. Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria. Scientific Reports. 2021. In press.