Confocal Imaging of Mitochondrial Movement within Oligodendrocytes in Organotypic Brain Slice Cultures

Begin with a confocal microscope bath chamber setup with a circulating imaging solution and controlled temperature.  

Take a hydrophilic polymeric membrane containing an organotypic mouse brain slice in which the oligodendrocytes have been transduced to express a mitochondria-specific red fluorescent protein and a cytosol-specific green fluorescent protein.

Dual fluorescence allows clear visualization of mitochondrial dynamics within the cytosol of the oligodendrocyte.

Submerge the slice in the center of the bath and secure it with a harp anchor.

Lower the objective and select a healthy oligodendrocyte for scanning.

Adjust laser power to minimize cell toxicity and photobleaching.

Set microscope parameters and then record time-lapse images of fluorescently labeled mitochondria.

Capture z-stack images at different depths to obtain a high-resolution, three-dimensional view of the entire cell.

Process the images to visualize mitochondrial movement within the oligodendrocyte.