MitoMoby Mitochondrial Morphology Analysis Software


MitoMoby is a high throughput and high content microscopic method for the analysis of mitochondrial morphology (MM) and membrane potential. MitoMoby delineates a procedure for MM analysis using the R platform for data analysis, annotation, and image registration and GE INCell Investigator (specifically, the GE INCell Developer Toolbox package) platform for image analysis.

Video Link: Quantitative Analysis of Mitochondrial Morphology

Seven measures of morphological analysis are reported in MitoMoby: total mitochondrial object area (comprised of all phenotypes, representative of total mitochondrial mass), puncta area, rod area, networked area, large & round area, total mitochondrial object count, and the average fiber length (length along all branches) of networked mitochondrial objects.

Analysis of mitochondrial morphology and membrane potential under experimental conditions is achieved via the following steps:

  1. Image acquisition via wide-field fluorescence microscopy (using the GE INCell Analyzer 2000 Instrument);

  2. Image analysis (using GE INCell Developer Toolbox) including:

    1. Preprocessing (2-D deconvolution and intensity normalization);

    2. Segmentation, (identification of mitochondrial objects);

    3. Classification into 4 morphological subtypes, (puncta, rods, networks, and large & round);

    4. Color definition using a machine learning-based classifier;

    5. Calculation of morphological measures such as area, count, and length;

    6. Quality control to eliminate small, dim objects

  3. Data annotation with experimental meta-data (treatment, concentration, plate, etc.) to facilitate unsupervised and supervised exclusion of low-quality cells and microscopic fields (out of focus, poorly stained) (using R and Excel);

  4. Data graphing and analysis (using R); and

  5. Statistical analysis (using R, Excel, and GraphPad).


This method may be used to diagnose mitochondrial diseases involving dysregulation of morphological machinery or mitochondrial function. It can also be used as a research tool to investigate the regulation of mitochondrial dynamics. Most suitably, researchers can use this tool to assess pharmacological/toxicological evaluation of compound libraries or evaluate mitochondrial health in a large patient sample.

The MitoMoby software suite includes a detailed user guide delineating steps from image acquisition to data graphing and analysis along with the relevant files.

MitoMoby is available for online licensing and download at .

Overview: The regulation of MM has been implicated as both a determinant and indicator of metabolic dysfunction in neurodegeneration and toxicology. Morphological binning is used to quantify mitochondrial interconnectedness. Distinct morphological categories of mitochondrial shapes can be individually analyzed, increasing the statistical power to quantify phenotypes. Additionally, mitochondrial inner membrane potential is analyzed in an automated fashion, versus total cell fluorescence.

Degenerative diseases are those in which the structure or function of the affected tissues or organs will progressively deteriorate over time. Some examples of degenerative disease include retinal degenerative disease, Amyotrophic Lateral Sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. It is commonly know that mitochondrial damage and/or dysfunction causes degenerative diseases. The mutation rates for mitochondria are very high due to the harmful free radicals that are released at byproducts from ATP production. The circulation of these free radicals leads to the activation of reactive nitrogen compounds, which in turn activates genes in DNA that are associated with many degenerative diseases. Free floating mitochondrial DNA (mtDNA) lacks protection within its own membrane, and is therefore easily damaged by the free radicals it produces.

Applications: Research, pharmacological/toxicological evaluation

Key Words: Mitochondria, mitochondrial morphology, morphological binning, mitochondrial membrane potential.

Inventors: Anthony Leonard, Craig Beeson, Baerbel Rohrer, Rick Schnellmann, Bethany Wolf, Jaime Lynn Speiser, Robert Cameron, Yuri Peterson

Publications: Leonard et al. "Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015. 1853:2.

Patent Status: Copyright

MUSC-FRD Technology ID: P13108 

Patent Information:
Research Tool
For Information, Contact:
Scott Davis
Associate Director, Licensing
MUSC Foundation for Research Development
Craig Beeson
Baerbel Rohrer
Anthony Leonard
Rick Schnellmann
Bethany Wolf
Jaime Speiser
Robert Cameron
Yuri Peterson
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