Application Note

Measuring mitochondrial shape and distribution using the ImageXpress Micro High-Content Imaging System

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The evaluation of mitochondrial responses is often used to determine the specific effect of chemical compounds on cell health. These responses may involve the ability of the mitochondria to sustain a steady membrane potential as exhibited by the intensity of fluorescent indicators such as some of the MitoTracker™ dyes. Other types of measurable responses are changes in the morphology and distribution of the organelle within the cell.

High-content imaging assays allow cells cultured and treated in microplates to be imaged alive without wash steps for realtime studies of cells in their most natural state, although certain dyes remain after fixation if an endpoint assay is desired. Combining high magnification, confocal optics, and collection of z-planes enables generation of 3D images for subsequent analysis as a 3D volume, vastly increasing the amount of information that can be obtained from every cell.

These assays can be performed in an automated screening environment with the ImageXpress® Micro Confocal High-Content Imaging System and MetaXpress® High-Content Image Acquistion and Analysis Software.


  • MitoTracker Orange CMTMRos (Life Technologies P/N M7510)
  • WI-38 human lung fibroblast cells (P12)
  • Rotenone
  • Chloroquine
  • 384-well black, clear-bottom microplates (BD Falcon P/N 353962)
  • Hoechst 33342 nuclear stain
  • ImageXpress Micro Confocal High-Content Imaging System

Assay method

WI-38 cells were plated at 7,500 cells per well in 50 μL per well in a 384-well tissue culture treated microplate. They were allowed to attach for 30 minutes in a 37°C, 5% CO2 incubator. Wells were then treated for 30 minutes with rotenone or chloroquine at 3 different concentrations. Finally, 10 μL of a 6X concentration of MitoTracker Orange + Hoechst was added to each well for a final concentration of 100 nM mitochondrial dye and 5 μM Hoechst. After 90 minutes incubation, plates were imaged on the ImageXpress Micro Confocal system using the DAPI and TRITC filter sets with a 40X or a 60X Plan Apo objective (Figure 1).


Figure 1. Raw 2D projection images taken with 60X objective. Mitochondria in cells treated with 50 μM Rotenone (top) and Chloroquine (bottom).

Data analysis

Cells imaged on the ImageXpress Micro Confocal system were visualized and analyzed in MetaXpress Software using the 3D analysis module. With step-wise user-input, the module accurately segments nuclei, outputs measurements on the total population of mitochondria in addition to classifying them as condensed or elongated, and supplies measurements on the separate mitochondrial morphologies. The 3D rendering from the z-series allows verification that the segmentation mask is accurately identifying objects in the raw images as well as highlighting morphometric and spatial parameters that may be of interest for analysis.


Figure 2. 3D rendering of isosurface overlays of stained mitochondria (red) and nuclei (white). (Left) 50 μM Rotenone-treated cells exhibit condensation and close proximity to the nuclei. (Right) Chloroquine-treated cells demonstrate elongated mitochondria spread throughout the cells.


This assay indicated that Chloroquine-treated cells had increased mitochondria volume while maintaining the ratio of elongated to condensed morphology found in control cells. Rotenone-treated cells exhibited condensation of mitochondria shown by a decrease in total volume as well as an increase in the ratio of condensed mitochondria compared to a control.

The ImageXpress Micro Confocal High-Content Imaging System used for acquiring high-quality images at multiple z-heights paired with the MetaXpress High-Content Image Acquisition and Analysis Software with 3D analysis module offers scientists a fast and robust image-based quantitation of multiple subcellular mitochondrial responses.


Figure 3. Measured parameters comparing cells treated with 50 μM compound to untreated controls.

Compatible with this Molecular Devices system


ImageXpress Micro Confocal High-Content Imaging System

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