This article describes a method for imaging mitochondrial dynamics in oligodendrocytes using confocal microscopy. The technique involves the use of dual fluorescence to visualize mitochondrial movement within the cytosol of the cells.
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.
Use forceps to transfer the confetti with the slice from the Petri dish to the top of the bath. Then place the two tips of the forceps on each side of the confetti so that they touch the confetti without touching the slice. And push the confetti through the solution to the bottom of the bath. Center the confetti in the middle of the bath.
Use forceps to place the hold-down anchor or harp on top of the confetti without touching the slice. Then turn the peristaltic pump back on. Lower the objective down to the sample. Then use the eyepieces and the fluorescent lamp to quickly find a healthy looking oligodendrocyte with fluorescent protein expression.
Use the live function in the software to identify the cell on the screen. And choose the area of interest. Here, an area containing several primary processes and one myelin sheath will be imaged. Zoom in on the field of interest. This will typically produce an image with pixel size of 0.14 to 0.30 microns.
Select the Regions tool and draw a rectangle around the area. Choose the option to scan only the selected region. Scanning time and thus laser exposure is reduced by only scanning the selected region.
Adjust the laser power and gain to get a good view of the mitochondria. Use low laser power and high gain to avoid bleaching during live imaging. If necessary, adjust the digital offset.
Minimize scanning time by selecting a fast scanning speed. Set the frequency and duration of timelapse recording for mitochondrial imaging and oligodendrocytes. Capture images every two seconds for a total of 20 minutes. Start the time lapse recording.
The continuous flow of solution can cause movement of the slice. It's worth spending some time minimizing this movement by adjusting the in and outlets of the bath. You can also reduce the pump speed.
Continually monitor the screen during time lapse imaging, as small changes in focus usually still occur. Adjust the focus as necessary. After time lapse imaging, capture a Z-stack of the whole cell, for future identification of the cell and imaged process.
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