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Microtubule associated protein light chain 3(LC3) is a ubiquitin-like protein that binds to autophagosomes (AVs). Cellular biologists transfect mammalian cells with GFP tagged LC3 to track and follow the fate of AVs in the cell and to measure autophagic flux. Also, GFP-LC3 puncta that colocalize with mitochondria is a way to measure the initiation of mitochondrial autophagy, the catabolic process by which mitochondria are targeted for lysosomal degradation.

Note: The macro can be used in three versions of Image J: 1.39, 1.44 and 1.46. The macro measures mitochondrial morphology, mitochondrial content (% cell area occupited by mitochondria), colocalization of GFP-LC3 puncta with mitochondria and cell morphology/area.

For specific instructions on how to analyze mitochondrial morphology please consult the “Mitochondrial Morphology” macro in this WIKI site.

There are two files that can be downloaded depending on the Image J version that the user wishes to use. Download the macro file that corresponds to the correct Image J version (1.39 and 1.44) below.

This macro is currently maintained and supported by a NIH P20GM103354-04 grant.


The macro was fully developed and approved for use by: Ruben Dagda, Ph.D. , Department of Pharmacology, University of Nevada School of Medicine. Email:

Contributor: Charleen Chu, University of Pittsburgh. Contributed to designing experimental methodology to validate the macro.


It is a multifunctional macro to do a variety of measurements at once: mitochondrial content, morphology, colocalization and mitochondrial autophagy. Macro allows you to measure the number of GFP-LC3 puncta (using the colocalization function of this macro), and GFP-LC3 puncta that colocalize with mitochondria, and can concurrently measure mitochondrial content and morphology in the same cell.

NOTE: This macro only works for RGB images. If black and white images are desired, then just delete the run(“RGB Split”); code lines in the macro, save it as a different version and re-run it. :-)


1. First, the macro allows the user time to select the region of interest or the cell to be analyzed using the polygon selection tool. The macro then splits the RGB image into individual channels (blue, green, and red), closes the blue and red channels, and extracts the red channel to grayscale followed by photographic inversion of the pixels. 2. The macro will allow the user time to assign the regions of interest to be measured by manually thresholding for minimal and maximal pixel values using the “Threshold” function under the Image pull-down menu. The macro then measures mitochondrial morphology (circularity and area/perimeter), mitochondrial content (percentage of cytosolic area occupied by mitochondria) and cellular area and perimeter. 3. Also, the user can analyze for the colocalization of GFP-LC3 puncta with mitochondria as an index of mitochondrial autophagy (mitophagy) by pressing the F12 key. The macro will output the total number and size of GFP-LC3 puncta (green channel), total mitochondrial particles (red channel) number of colocalized particles (RGB image) using the colocalization function of Image J.

Installation and Use

Download from here for Image J version 1.39: or copy the code of this macro, paste it (Notepad for PC Users or Word document for Mac users)and save it as a text file. Move the text file on to the “Macros” folder, install the macro and press “F9” to start the macro.


Then press F10 to process the image.

Multiple macros within this macro were added:

a) Press Function 9 to start the macro and locate the RGB image. Select the area of interest and then press F10 key to start the Mitophagy macro and process the image of interest. Open up the RGB file to be analyzed and select the cell to be analyzed.

b) Then press Function 11 key to invoke the “Analyze Images” macro in order to process the RGB image by inverting the pixels and for thresholding the mitochondrial pixels. The macro will do the rest of the analysis for you by analyzing the mitochondrial morphology (circularity, area/perimeter ratio) and content (expressed as percentage of total cytosolic area occupied by mitochondrial pixels)

c) Press Function F12 if you want to analyze the colocalization of GFP-LC3 puncta with mitochondria. Results are shown as total number of GFP-LC3 puncta that colocalize with mitochondria. Results are displayed under the “Log” window. You may want to calibrate and optimize the sensitivity of the “Colocalization” plug-in for images of the red and green channels by playing with the threshold percentages for the ratio, thresholds for channel 1 and 2. So far, a ratio of 85% has worked the best for high quality confocal RGB images taken at a magnification of 60X with a zoom factor ratio 4.0 The macro will output the total number and size of GFP-LC3 puncta (green channel), total mitochondrial particles (red channel) number of colocalized particles (RGB image) using the colocalization function of Image J.

4) Press Function 4 key if you want to close any unnecessary windows at any time.

You can measure as many cells as necessary within the same RGB image by simply re-tracing another cell and pressing the F10 function to process the image and F11 to measure mitochondrial content and morphology.


Download the macro by clicking on the following link here: mitophagy_ver_1_44_final_colocalization_f.txt


The authors of the macro reserve the copyrights of the original macro. However, you are welcome to distribute, modify and use the program under the terms of the GNU General Public License as stated here: ( as long as you attribute proper acknowledgement to the authors as mentioned above.

If publishing data analyzed employing this macro or any derivative thereof, please cite the following papers:


1. Dagda, R.K., Zhu, J., Kulich, S.M., and Chu, C.T. Mitochondrially localized ERK2regulates mitophagy and autophagic cell stress. Autophagy. 4 (6): 770-82, 2008. PMID: 18594198

2. Zhu J, Dagda RK, Chu CT. Monitoring mitophagy in neuronal cell cultures. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Methods Mol Biol. 2011;793:325-39, 2011


Function added to close any unnecessary windows by pressing the [F4] key.

Known Bugs

This macro only works on the old version of Image J, earlier versions than that of Image J 1.42 The circularity and minor axis have been included in the mitochondrial morphology function of this macro. These are included in the output or results of the analysis as “mitochondrial morphology based on circ.” and mitochondrial morphology based on minor axis“. These additional quantifications helps to distinguish swollen mitochondria from elongated mitochondria. Please direct questions to Ruben K. Dagda at:

macro/mitpophagy_mitochondrial_morphology_content_lc3_colocalization_macro.txt · Last modified: 2019/04/12 13:13 by

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