Mitochondrial imaging (shape/volume/number/fusion/fission events)
Fluorescent acquisition and treatment
Mitochondrial dynamics imaging
Developments in imaging technology and fluorescent probes have made it possible to visualize cellular and subcellular dynamics in space and time. To investigate mitochondrial dynamics in patient fibroblasts, we used an inverted wide-field Leica video-microscope (DMI 6000 B, Leica Microsystems, Wetzlar, Germany) equipped with a Roper CoolSNAP HQ2 (Photometrics, Tucson, AZ, USA), a high-sensitivity CCD camera for quantitative fluorescence microscopy. The imaging system driven by MetaMorph® (Molecular Devices, Sunnyvale, CA, USA) constitutes a system for multi-dimensional image acquisition. A piezoelectric driver mounted underneath the objective lens allows faster Z-step movements, keeping the sample immobile while shifting the objective lens.
We use image restoration algorithms to reassign the optical blur to its original location (Huygens software, Scientific Volume Imaging, Hilversum, The Netherlands).
We first use the Metamorph® software for image processing, morphometry and mitochondrial measurements of motility (Chevrollier et al., 2008; Cassereau et al. 2009). By using Imaris 7.1.1® software (Bitplane, Zurich, Switzerland) we increase the analysis level by a 3D processing.This technique allows the determination of mitochondrial length, volume, number of connections per cell, the nucleoids distribution within the cells and the mitochondrial membrane potential. Using this technology we showed altered mitochondrial network structure in the fibroblasts carrying mutations in OPA1, MFN2, and GDAP1 in comparison to controls. Most of the OPA1 fibroblasts showed mitochondrial fragmentation whereas MFN2 showed highly denses and interconnected mitochondrial network.
Time lapse acquisition were performed to show mitochondrial fusion or fission events (mov 1).